U.S. patent application number 13/916129 was filed with the patent office on 2013-12-26 for antigen binding proteins that bind igf1r.
The applicant listed for this patent is Sorrento Therapeutics Inc.. Invention is credited to Randy Gastwirt, Guodi Lu, Heyue Zhou.
Application Number | 20130344069 13/916129 |
Document ID | / |
Family ID | 49769658 |
Filed Date | 2013-12-26 |
United States Patent
Application |
20130344069 |
Kind Code |
A1 |
Gastwirt; Randy ; et
al. |
December 26, 2013 |
Antigen Binding Proteins that Bind IGF1R
Abstract
There is disclosed compositions and methods relating to or
derived from anti-IGF1R antibodies. More specifically, there is
disclosed fully human antibodies that bind IGF1R, IGF1R-binding
fragments and derivatives of such antibodies, and IGF1R-binding
polypeptides comprising such fragments. Further still, there is
disclosed nucleic acids encoding such antibodies, antibody
fragments and derivatives and polypeptides, cells comprising such
polynucleotides, methods of making such antibodies, antibody
fragments and derivatives and polypeptides, and methods of using
such antibodies, antibody fragments and derivatives and
polypeptides, including methods of treating or diagnosing subjects
having IGF1R related disorders or conditions, including various
inflammatory disorders and various cancers.
Inventors: |
Gastwirt; Randy; (San Diego,
CA) ; Zhou; Heyue; (San Diego, CA) ; Lu;
Guodi; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sorrento Therapeutics Inc. |
San Diego |
CA |
US |
|
|
Family ID: |
49769658 |
Appl. No.: |
13/916129 |
Filed: |
June 12, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61662905 |
Jun 21, 2012 |
|
|
|
Current U.S.
Class: |
424/135.1 ;
424/174.1; 530/387.3; 530/389.7 |
Current CPC
Class: |
A61P 35/00 20180101;
C07K 16/30 20130101; C07K 16/2863 20130101; C07K 2317/73 20130101;
A61P 35/02 20180101; C07K 2317/92 20130101; C07K 2317/76
20130101 |
Class at
Publication: |
424/135.1 ;
530/389.7; 530/387.3; 424/174.1 |
International
Class: |
C07K 16/30 20060101
C07K016/30 |
Claims
1. A fully human antibody of an IgG class that binds to an IGF1R
epitope with a binding affinity of at least 10.sup.-6M, that has a
heavy chain variable domain sequence that is at least 95% identical
to the amino acid sequences selected from the group consisting of
SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO.
9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ
ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO.
27, SEQ ID NO. 29, SEQ ID NO. 31, and combinations thereof, and
that has a light chain variable domain sequence that is at least
95% identical to the amino acid sequences selected from the group
consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO.
8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ
ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO.
26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, and combinations
thereof.
2. The fully human antibody of claim 1, wherein the antibody has a
heavy chain/light chain variable domain sequence selected from the
group consisting of SEQ ID NO. 1/SEQ ID NO. 2 (called GFA1 herein),
SEQ ID NO. 3/SEQ ID NO. 4 (called GFA3 herein), SEQ ID NO. 5/SEQ ID
NO. 6 (called GFA5 herein), SEQ ID NO. 7/SEQ ID NO. 8 (called GFA6
herein), SEQ ID NO. 9/SEQ ID NO. 10 (called GFA12 herein), SEQ ID
NO. 11/SEQ ID NO. 12 (called GFC2 herein), SEQ ID NO. 13/SEQ ID NO.
14 (called A2 herein), SEQ ID NO. 15/SEQ ID NO. 16 (called A11
herein), SEQ ID NO. 17/SEQ ID NO. 18 (called B9 herein), SEQ ID NO.
19/SEQ ID NO. 20 (called B10 herein), SEQ ID NO. 21/SEQ ID NO. 22
(called A6 herein), SEQ ID NO. 23/SEQ ID NO. 24 (called C8 herein),
SEQ ID NO. 25/SEQ ID NO. 26 (called C4 herein), SEQ ID NO. 27/SEQ
ID NO. 28 (called E2 herein), SEQ ID NO. 29/SEQ ID NO. 30 (called
B3 herein), SEQ ID NO. 31/SEQ ID NO. 32 (called D12 herein), and
combinations thereof.
3. A fully human Fab antibody fragment, having a variable domain
region from a heavy chain and a variable domain region from a light
chain, wherein the heavy chain variable domain sequence that is at
least 95% identical to the amino acid sequences selected from the
group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ
ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO.
15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ
ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, and
combinations thereof, and that has a light chain variable domain
sequence that is at least 95% identical to the amino acid sequences
selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4,
SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID
NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22,
SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID
NO. 32, and combinations thereof.
4. The fully human antibody Fab fragment of claim 3, wherein the
antibody has a heavy chain/light chain variable domain sequence
selected from the group consisting of SEQ ID NO. 1/SEQ ID NO. 2,
SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO.
7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID
NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16,
SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID
NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO.
25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID
NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, and combinations thereof.
5. A single chain human antibody, having a variable domain region
from a heavy chain and a variable domain region from a light chain
and a peptide linker connection the heavy chain and light chain
variable domain regions, wherein the heavy chain variable domain
sequence that is at least 95% identical to the amino acid sequences
selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3,
SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO.
13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ
ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO.
31, and combinations thereof, and that has a light chain variable
domain sequence that is at least 95% identical to the amino acid
sequences selected from the group consisting of SEQ ID NO. 2, SEQ
ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12,
SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID
NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30,
SEQ ID NO. 32, and combinations thereof.
6. The fully human single chain antibody of claim 5, wherein the
single chain fully human antibody has a heavy chain/light chain
variable domain sequence selected from the group consisting of SEQ
ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ
ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10,
SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID
NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO.
19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID
NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28,
SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, and
combinations thereof.
7. The fully human single chain antibody of claim 5, wherein the
fully human single chain antibody has both a heavy chain variable
domain region and a light chain variable domain region, wherein the
single chain fully human antibody has a heavy chain/light chain
variable domain sequence selected from the group consisting of SEQ
ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ
ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10,
SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID
NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO.
19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID
NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28,
SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, and
combinations thereof.
8. A method for treating a broad spectrum of mammalian cancers,
comprising administering an effective amount of an anti-IGF1R
polypeptide, wherein the anti-IGF1R polypeptide is selected from
the group consisting of a fully human antibody of an IgG class that
binds to a IGF1R epitope with a binding affinity of at least
10.sup.-6 M, a fully human Fab antibody fragment, having a variable
domain region from a heavy chain and a variable domain region from
a light chain, a single chain human antibody, having a variable
domain region from a heavy chain and a variable domain region from
a light chain and a peptide linker connection the heavy chain and
light chain variable domain regions, and combinations thereof;
wherein the fully human antibody has a heavy chain variable domain
sequence that is at least 95% identical to the amino acid sequences
selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3,
SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO.
13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ
ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO.
31, and combinations thereof, and that has a light chain variable
domain sequence that is at least 95% identical to the amino acid
sequences selected from the group consisting of SEQ ID NO. 2, SEQ
ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12,
SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID
NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30,
SEQ ID NO. 32, and combinations thereof; wherein the fully human
Fab antibody fragment has the heavy chain variable domain sequence
that is at least 95% identical to the amino acid sequences selected
from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO.
5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID
NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23,
SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, and
combinations thereof, and that has the light chain variable domain
sequence that is at least 95% identical to the amino acid sequences
selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4,
SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID
NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22,
SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID
NO. 32, SEQ ID NO. 34, and combinations thereof; and wherein the
single chain human antibody has the heavy chain variable domain
sequence that is at least 95% identical to the amino acid sequences
selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3,
SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO.
13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ
ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO.
31, and combinations thereof, and that has the light chain variable
domain sequence that is at least 95% identical to the amino acid
sequences selected from the group consisting of SEQ ID NO. 2, SEQ
ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12,
SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID
NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30,
SEQ ID NO. 32, and combinations thereof.
9. The method for treating a broad spectrum of mammalian cancers of
claim 8, wherein the fully human antibody has a heavy chain/light
chain variable domain sequence selected from the group consisting
of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO.
5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO.
10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ
ID NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO.
19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID
NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28,
SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, and
combinations thereof.
10. The method for treating a broad spectrum of mammalian cancers
of claim 8, wherein the fully human antibody Fab fragment has both
a heavy chain variable domain region and a light chain variable
domain region wherein the antibody has a heavy chain/light chain
variable domain sequence selected from the group consisting of SEQ
ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ
ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10,
SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID
NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO.
19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID
NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28,
SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, and
combinations thereof.
11. The method for treating a broad spectrum of mammalian cancers
of claim 8, wherein the fully human single chain antibody has both
a heavy chain variable domain region and a light chain variable
domain region, wherein the single chain fully human antibody has a
heavy chain/light chain variable domain sequence selected from the
group consisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID
NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID
NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ
ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO.
18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ
ID NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO.
27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID
NO. 32, and combinations thereof.
12. The method for treating a broad spectrum of mammalian cancers
of claim 8, wherein the broad spectrum of mammalian cancers to be
treated is selected from the group consisting of the osteosarcoma,
rhabdomyosarcoma, neuroblastoma, any pediatric cancer, kidney
cancer, leukemia, renal transitional cell cancer, Werner-Morrison
syndrome, acromegaly, bladder cancer, Wilm's cancer, ovarian
cancer, pancreatic cancer, benign prostatic hyperplasia, breast
cancer, prostate cancer, bone cancer, lung cancer, gastric cancer,
colorectal cancer, cervical cancer, synovial sarcoma, diarrhea
associated with metastatic carcinoid, vasoactive intestinal peptide
secreting tumors, head and neck cancer, squamous cell carcinoma,
multiple myeloma, solitary plasmacytoma, renal cell cancer,
retinoblastoma, germ cell tumors, hepatoblastoma, hepatocellular
carcinoma, melanoma, rhabdoid tumor of the kidney, Ewing Sarcoma,
chondrosarcoma, haemotological malignancy, chronic lymphoblastic
leukemia, chronic myelomonocytic leukemia, acute lymphoblastic
leukemia, acute lymphocytic leukemia, acute myelogenous leukemia,
acute myeloblastic leukemia, chronic myeloblastic leukemia,
Hodgkin's disease, non-Hodgkin's lymphoma, chronic lymphocytic
leukemia, chronic myelogenous leukemia, myelodysplastic syndrome,
hairy cell leukemia, mast cell leukemia, mast cell neoplasm,
follicular lymphoma, diffuse large cell lymphoma, mantle cell
lymphoma, Burkitt Lymphoma, mycosis fungoides, seary syndrome,
cutaneous T-cell lymphoma, chronic myeloproliferative disorders, a
central nervous system tumor, brain cancer, glioblastoma,
non-glioblastoma brain cancer, meningioma, pituitary adenoma,
vestibular schwannoma, a primitive neuroectodermal tumor,
medulloblastoma, astrocytoma, anaplastic astrocytoma,
oligodendroglioma, ependymoma and choroid plexus papilloma, a
myeloproliferative disorder, polycythemia vera, thrombocythemia,
idiopathic myelfibrosis, soft tissue sarcoma, thyroid cancer,
endometrial cancer, carcinoid cancer, germ cell tumors, liver
cancer, Grave's disease, and combinations thereof.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This patent application claims priority to U.S. provisional
patent application 61/662,905 filed 21 Jun. 2012.
TECHNICAL FIELD
[0002] The present disclosure provides compositions and methods
relating to or derived from anti-IGF1R antibodies. More
specifically, the present disclosure provides human antibodies that
bind IGF1R, IGF1R-binding fragments and derivatives of such
antibodies, and IGF1R-binding polypeptides comprising such
fragments. Further still, the present disclosure provides nucleic
acids encoding such antibodies, antibody fragments and derivatives
and polypeptides, cells comprising such polynucleotides, methods of
making such antibodies, antibody fragments and derivatives and
polypeptides, and methods of using such antibodies, antibody
fragments and derivatives and polypeptides, including methods of
treating or diagnosing subjects having IGF1R related disorders or
conditions, including various inflammatory disorders and various
cancers.
BACKGROUND
[0003] The insulin-like growth factors, also known as somatomedins,
include insulin-like growth factor-I (IGF-I) and insulin-like
growth factor-II (IGF-II) (Klapper, et al., (1983) Endocrinol.
112:2215 and Rinderknecht, et al., (1978) Febs. Lett. 89:283).
These growth factors exert mitogenic activity on various cell
types, including tumor cells (Macaulay, (1992) Br. J. Cancer
65:311), by binding to a common receptor named the insulin-like
growth factor receptor-1 (IGF1R) (Sepp-Lorenzino, (1998) Breast
Cancer Research and Treatment 47:235). Interaction of IGFs with
IGF1R activates the receptor by triggering autophosphorylation of
the receptor on tyrosine residues (Butler, et al., (1998)
Comparative Biochemistry and Physiology 121:19). Once activated,
IGF1R, in turn, phosphorylates intracellular targets to activate
cellular signaling pathways. This receptor activation is critical
for stimulation of tumor cell growth and survival. Therefore,
inhibition of IGF1R activity represents a valuable potential method
to treat or prevent growth of human cancers and other proliferative
diseases.
[0004] Several lines of evidence indicate that IGF-I, IGF-II and
their receptor IGF1R are important mediators of the malignant
phenotype. Plasma levels of IGF-I have been found to be the
strongest predictor of prostate cancer risk (Chan, et al., (1998)
Science 279:563) and similar epidemiological studies strongly link
plasma IGF-I levels with breast, colon and lung cancer risk.
[0005] Overexpression of Insulin-like Growth Factor Receptor-1 has
also been demonstrated in several cancer cell lines and tumor
tissues. IGF1R is overexpressed in 40% of all breast cancer cell
lines (Pandini, et al., (1999) Cancer Res. 5:1935) and in 15% of
lung cancer cell lines. In breast cancer tumor tissue, IGF1R is
overexpressed 6-14 fold and IGF1R exhibits 2-4 fold higher kinase
activity as compared to normal tissue (Webster, et al., (1996)
Cancer Res. 56:2781 and Pekonen, et al., (1998) Cancer Res.
48:1343). Ninety percent of colorectal cancer tissue biopsies
exhibit elevated IGF1R levels wherein the extent of IGF1R
expression is correlated with the severity of the disease. Analysis
of primary cervical cancer cell cultures and cervical cancer cell
lines revealed 3- and 5-fold overexpression of IGF1R, respectively,
as compared to normal ectocervical cells (Steller, et al., (1996)
Cancer Res. 56:1762). Expression of IGF1R in synovial sarcoma cells
also correlated with an aggressive phenotype (i.e., metastasis and
high rate of proliferation; Xie, et al., (1999) Cancer Res.
59:3588). Furthermore, acromegaly, a slowly developing disease, is
caused by hypersecretion of growth hormone and IGF-I (Ben-Schlomo,
et al., (2001) Endocrin. Metab. Clin. North. Am. 30:565-583).
Antagonism of IGF1R function may be helpful in treating the
disease. There remains a need in the art for IGF1R antagonist
therapies for treating or preventing such disease and disorders. Of
particular utility are anti-IGF1R antibody based therapies.
SUMMARY
[0006] The present disclosure provides a fully human antibody of an
IgG class that binds to a IGF1R epitope with a binding affinity of
at least 10.sup.-6M, which has a heavy chain variable domain
sequence that is at least 95% identical to the amino acid sequences
selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3,
SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO.
13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ
ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO.
31, and combinations thereof, and that has a light chain variable
domain sequence that is at least 95% identical to the amino acid
sequences selected from the group consisting of SEQ ID NO. 2, SEQ
ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12,
SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID
NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30,
SEQ ID NO. 32, and combinations thereof. Preferably, the fully
human antibody has both a heavy chain and a light chain wherein the
antibody has a heavy chain/light chain variable domain sequence
selected from the group consisting of SEQ ID NO. 1/SEQ ID NO. 2
(called GFA1 herein), SEQ ID NO. 3/SEQ ID NO. 4 (called GFA3
herein), SEQ ID NO. 5/SEQ ID NO. 6 (called GFA5 herein), SEQ ID NO.
7/SEQ ID NO. 8 (called GFA6 herein), SEQ ID NO. 9/SEQ ID NO. 10
(called GFA12 herein), SEQ ID NO. 11/SEQ ID NO. 12 (called GFC2
herein), SEQ ID NO. 13/SEQ ID NO. 14 (called A2 herein), SEQ ID NO.
15/SEQ ID NO. 16 (called A11 herein), SEQ ID NO. 17/SEQ ID NO. 18
(called B9 herein), SEQ ID NO. 19/SEQ ID NO. 20 (called B10
herein), SEQ ID NO. 21/SEQ ID NO. 22 (called A6 herein), SEQ ID NO.
23/SEQ ID NO. 24 (called C8 herein), SEQ ID NO. 25/SEQ ID NO. 26
(called C4 herein), SEQ ID NO. 27/SEQ ID NO. 28 (called E2 herein),
SEQ ID NO. 29/SEQ ID NO. 30 (called B3 herein), SEQ ID NO. 31/SEQ
ID NO. 32 (called D12 herein), and combinations thereof.
[0007] The present disclosure provides a fully human Fab antibody
fragment, having a variable domain region from a heavy chain and a
variable domain region from a light chain, wherein the heavy chain
variable domain sequence that is at least 95% identical to the
amino acid sequences selected from the group consisting of SEQ ID
NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ
ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO.
19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ
ID NO. 29, SEQ ID NO. 31, and combinations thereof, and that has a
light chain variable domain sequence that is at least 95% identical
to the amino acid sequences selected from the group consisting of
SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO.
10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ
ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO.
28, SEQ ID NO. 30, SEQ ID NO. 32, and combinations thereof.
Preferably, the fully human antibody Fab fragment has both a heavy
chain variable domain region and a light chain variable domain
region wherein the antibody has a heavy chain/light chain variable
domain sequence selected from the group consisting of SEQ ID NO.
1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO.
6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID
NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO.
15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID
NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID NO. 24,
SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID
NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, and combinations
thereof.
[0008] The present disclosure provides a single chain human
antibody, having a variable domain region from a heavy chain and a
variable domain region from a light chain and a peptide linker
connection the heavy chain and light chain variable domain regions,
wherein the heavy chain variable domain sequence that is at least
95% identical to the amino acid sequences selected from the group
consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO.
7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ
ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO.
25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, and combinations
thereof, and that has a light chain variable domain sequence that
is at least 95% identical to the amino acid sequences selected from
the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6,
SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID
NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24,
SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, and
combinations thereof. Preferably, the fully human single chain
antibody has both a heavy chain variable domain region and a light
chain variable domain region, wherein the single chain fully human
antibody has a heavy chain/light chain variable domain sequence
selected from the group consisting of SEQ ID NO. 1/SEQ ID NO. 2,
SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO.
7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID
NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16,
SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID
NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO.
25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID
NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, and combinations thereof.
[0009] The present disclosure further provides a method for
treating a broad spectrum of mammalian cancers or a broad-spectrum
of inflammatory diseases and autoimmune diseases, comprising
administering an effective amount of an anti-IGF1R polypeptide,
wherein the anti-IGF1R polypeptide is selected from the group
consisting of a fully human antibody of an IgG class that binds to
a IGF1R epitope with a binding affinity of at least 10.sup.-6M, a
fully human Fab antibody fragment, having a variable domain region
from a heavy chain and a variable domain region from a light chain,
a single chain human antibody, having a variable domain region from
a heavy chain and a variable domain region from a light chain and a
peptide linker connection the heavy chain and light chain variable
domain regions, and combinations thereof;
[0010] wherein the fully human antibody has a heavy chain variable
domain sequence that is at least 95% identical to the amino acid
sequences selected from the group consisting of SEQ ID NO. 1, SEQ
ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11,
SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID
NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29,
SEQ ID NO. 31 and combinations thereof, and that has a light chain
variable domain sequence that is at least 95% identical to the
amino acid sequences selected from the group consisting of SEQ ID
NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ
ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO.
20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ
ID NO. 30, SEQ ID NO. 32, and combinations thereof;
[0011] wherein the fully human Fab antibody fragment has the heavy
chain variable domain sequence that is at least 95% identical to
the amino acid sequences selected from the group consisting of SEQ
ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9,
SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID
NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27,
SEQ ID NO. 29, SEQ ID NO. 31, and combinations thereof, and that
has the light chain variable domain sequence that is at least 95%
identical to the amino acid sequences selected from the group
consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO.
8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ
ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO.
26, SEQ ID NO. 28, SEQ ID NO. 30, and combinations thereof; and
[0012] wherein the single chain human antibody has the heavy chain
variable domain sequence that is at least 95% identical to the
amino acid sequences selected from the group consisting of SEQ ID
NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ
ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO.
19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ
ID NO. 29, SEQ ID NO. 31, and combinations thereof, and that has
the light chain variable domain sequence that is at least 95%
identical to the amino acid sequences selected from the group
consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO.
8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ
ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO.
26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, and combinations
thereof.
[0013] Preferably, the fully human antibody has both a heavy chain
and a light chain wherein the antibody has a heavy chain/light
chain variable domain sequence selected from the group consisting
of SEQ ID NO. 1/SEQ ID NO. 2 (called GFA1 herein), SEQ ID NO. 3/SEQ
ID NO. 4 (called GFA3 herein), SEQ ID NO. 5/SEQ ID NO. 6 (called
GFA5 herein), SEQ ID NO. 7/SEQ ID NO. 8 (called GFA6 herein), SEQ
ID NO. 9/SEQ ID NO. 10 (called GFA12 herein), SEQ ID NO. 11/SEQ ID
NO. 12 (called GFC2 herein), SEQ ID NO. 13/SEQ ID NO. 14 (called A2
herein), SEQ ID NO. 15/SEQ ID NO. 16 (called A11 herein), SEQ ID
NO. 17/SEQ ID NO. 18 (called B9 herein), SEQ ID NO. 19/SEQ ID NO.
20 (called B10 herein), SEQ ID NO. 21/SEQ ID NO. 22 (called A6
herein), SEQ ID NO. 23/SEQ ID NO. 24 (called C8 herein), SEQ ID NO.
25/SEQ ID NO. 26 (called C4 herein), SEQ ID NO. 27/SEQ ID NO. 28
(called E2 herein), SEQ ID NO. 29/SEQ ID NO. 30 (called B3 herein),
SEQ ID NO. 31/SEQ ID NO. 32 (called D12 herein), and combinations
thereof. Preferably, the fully human antibody Fab fragment has both
a heavy chain variable domain region and a light chain variable
domain region wherein the antibody has a heavy chain/light chain
variable domain sequence selected from the group consisting of SEQ
ID NO. 1/SEQ ID NO. 2 (called GFA1 herein), SEQ ID NO. 3/SEQ ID NO.
4 (called GFA3 herein), SEQ ID NO. 5/SEQ ID NO. 6 (called GFA5
herein), SEQ ID NO. 7/SEQ ID NO. 8 (called GFA6 herein), SEQ ID NO.
9/SEQ ID NO. 10 (called GFA12 herein), SEQ ID NO. 11/SEQ ID NO. 12
(called GFC2 herein), SEQ ID NO. 13/SEQ ID NO. 14 (called A2
herein), SEQ ID NO. 15/SEQ ID NO. 16 (called A11 herein), SEQ ID
NO. 17/SEQ ID NO. 18 (called B9 herein), SEQ ID NO. 19/SEQ ID NO.
20 (called B10 herein), SEQ ID NO. 21/SEQ ID NO. 22 (called A6
herein), SEQ ID NO. 23/SEQ ID NO. 24 (called C8 herein), SEQ ID NO.
25/SEQ ID NO. 26 (called C4 herein), SEQ ID NO. 27/SEQ ID NO. 28
(called E2 herein), SEQ ID NO. 29/SEQ ID NO. 30 (called B3 herein),
SEQ ID NO. 31/SEQ ID NO. 32 (called D12 herein), and combinations
thereof. Preferably, the fully human single chain antibody has both
a heavy chain variable domain region and a light chain variable
domain region, wherein the single chain fully human antibody has a
heavy chain/light chain variable domain sequence selected from the
group consisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID
NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID
NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ
ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO.
18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ
ID NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO.
27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID
NO. 32, and combinations thereof.
[0014] Preferably, the broad spectrum of mammalian cancers to be
treated is selected from the group consisting of the osteosarcoma,
rhabdomyosarcoma, neuroblastoma, any pediatric cancer, kidney
cancer, leukemia, renal transitional cell cancer, Werner-Morrison
syndrome, bladder cancer, Wilm's cancer, ovarian cancer, pancreatic
cancer, benign prostatic hyperplasia, breast cancer, prostate
cancer, bone cancer, lung cancer, gastric cancer, colorectal
cancer, cervical cancer, synovial sarcoma, diarrhea associated with
metastatic carcinoid, vasoactive intestinal peptide secreting
tumors, head and neck cancer, squamous cell carcinoma, multiple
myeloma, solitary plasmacytoma, renal cell cancer, retinoblastoma,
germ cell tumors, hepatoblastoma, hepatocellular carcinoma,
melanoma, rhabdoid tumor of the kidney, Ewing Sarcoma,
chondrosarcoma, haemotological malignancy, chronic lymphoblastic
leukemia, chronic myelomonocytic leukemia, acute lymphoblastic
leukemia, acute lymphocytic leukemia, acute myelogenous leukemia,
acute myeloblastic leukemia, chronic myeloblastic leukemia,
Hodgkin's disease, non-Hodgkin's lymphoma, chronic lymphocytic
leukemia, chronic myelogenous leukemia, myelodysplastic syndrome,
hairy cell leukemia, mast cell leukemia, mast cell neoplasm,
follicular lymphoma, diffuse large cell lymphoma, mantle cell
lymphoma, Burkitt Lymphoma, mycosis fungoides, seary syndrome,
cutaneous T-cell lymphoma, chronic myeloproliferative disorders, a
central nervous system tumor, brain cancer, glioblastoma,
non-glioblastoma brain cancer, meningioma, pituitary adenoma,
vestibular schwannoma, a primitive neuroectodermal tumor,
medulloblastoma, astrocytoma, anaplastic astrocytoma,
oligodendroglioma, ependymoma and choroid plexus papilloma, a
myeloproliferative disorder, polycythemia vera, thrombocythemia,
idiopathic myelfibrosis, soft tissue sarcoma, thyroid cancer,
endometrial cancer, carcinoid cancer, germ cell tumors, liver
cancer, and combinations thereof.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1 shows the affinity determination of the anti-IGF1R
antibody C2 determined by two methods. The sensor was coated with
either rhIGF1R or C2 IgG and then incubated with the other to
determine affinity using the Forte Bio Octet Red Machine. The
affinity of C2 was determined to be 5.38 nM or 1.45 nM using the
two methods.
[0016] FIG. 2 shows the affinity determination of anti-IGF1R
antibody B9 on anti-hIgG Fc capture sensor using the Forte Bio
Octet Red Machine. The affinity of B9 was determined to be 1.25
nM.
[0017] FIG. 3 shows the cell binding and the EC50 values for cell
binding to MCF7 cells of the anti-IGF1R antibodies.
[0018] FIG. 4 shows the results of an ELISA assay comparing the
binding of anti-IGF1R antibodies to IGF1R and the Insulin Receptor.
Clones B9, B10, and C8 do not cross-react with the Insulin
Receptor.
[0019] FIG. 5 shows IGF1-stimulated, auto-phosphorylation of IGF1R
in MCF7 breast cancer cells. Various anti-IGF1R antibodies were
compared at an antibody concentration of 10 .mu.g/ml and clones A6,
B9, B10, B10VAR, C2 and C8 show the greatest antagonism.
[0020] FIG. 6 shows the IC.sub.50 values for the inhibition of
IGF1-stimulated IGF1R auto-phosphorylation in MCF7 breast cancer
cells for anti-IGF1R antibody clones A6, B9, B10VAR, C2 and C8. B9
shows superior antagonism of IGF1R auto-phosphorylation with an
IC.sub.50 of 94 .mu.M.
[0021] FIG. 7 shows the inhibition of IGF1-stimulated proliferation
in MCF7 cells by the anti-IGF1R antibodies. Compared to cells not
treated with antibody, anti-IGF1R antibody clones C2, B10VAR, and
C8 show strong dose-dependent antagonism of IGF1-stimulated
proliferation.
[0022] FIG. 8 shows that MCF7 cells treated with 100 ng/ml IGF2
showed robust activating phosphorylation of IGF1R (column 2, IGF2
Alone, compared to column 1, Untreated). Pre-treatment of cells
with anti-IGF1R antibodies variably blocked this activation of
IGF1R. Clone B10 showed the most potent antagonism of IGF1R
auto-phosphorylation. Data are shown as absorption at 450 nm (ABS
450 nm) of triplicate samples +/- Std Error and were directly
proportional to IGF1R phosphorylation/activation.
DETAILED DESCRIPTION
[0023] The present disclosure provides a fully human antibody of an
IgG class that binds to a IGF1R epitope with a binding affinity of
10.sup.-6M or less, that has a heavy chain variable domain sequence
that is at least 95% identical to the amino acid sequences selected
from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO.
5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID
NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23,
SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, and
combinations thereof, and that has a light chain variable domain
sequence that is at least 95% identical to the amino acid sequences
selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4,
SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID
NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22,
SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID
NO. 32, and combinations thereof. Preferably, the fully human
antibody has both a heavy chain and a light chain wherein the
antibody has a heavy chain/light chain variable domain sequence
selected from the group consisting of SEQ ID NO. 1/SEQ ID NO. 2
(called GFA1 herein), SEQ ID NO. 3/SEQ ID NO. 4 (called GFA3
herein), SEQ ID NO. 5/SEQ ID NO. 6 (called GFA5 herein), SEQ ID NO.
7/SEQ ID NO. 8 (called GFA6 herein), SEQ ID NO. 9/SEQ ID NO. 10
(called GFA12 herein), SEQ ID NO. 11/SEQ ID NO. 12 (called GFC2
herein), SEQ ID NO. 13/SEQ ID NO. 14 (called A2 herein), SEQ ID NO.
15/SEQ ID NO. 16 (called A11 herein), SEQ ID NO. 17/SEQ ID NO. 18
(called B9 herein), SEQ ID NO. 19/SEQ ID NO. 20 (called B10
herein), SEQ ID NO. 21/SEQ ID NO. 22 (called A6 herein), SEQ ID NO.
23/SEQ ID NO. 24 (called C8 herein), SEQ ID NO. 25/SEQ ID NO. 26
(called C4 herein), SEQ ID NO. 27/SEQ ID NO. 28 (called E2 herein),
SEQ ID NO. 29/SEQ ID NO. 30 (called B3 herein), SEQ ID NO. 31/SEQ
ID NO. 32 (called D12 herein), and combinations thereof.
[0024] The present disclosure provides a fully human Fab antibody
fragment, having a variable domain region from a heavy chain and a
variable domain region from a light chain, wherein the heavy chain
variable domain sequence that is at least 95% identical to the
amino acid sequences selected from the group consisting of SEQ ID
NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ
ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO.
19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ
ID NO. 29, SEQ ID NO. 31, and combinations thereof, and that has a
light chain variable domain sequence that is at least 95% identical
to the amino acid sequences selected from the group consisting of
SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO.
10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ
ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO.
28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 34, SEQ ID NO. 36, SEQ
ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO.
46, and combinations thereof. Preferably, the fully human antibody
Fab fragment has both a heavy chain variable domain region and a
light chain variable domain region wherein the antibody has a heavy
chain/light chain variable domain sequence selected from the group
consisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4,
SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO.
9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID
NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18,
SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID
NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO.
27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID
NO. 32, and combinations thereof.
[0025] The present disclosure provides a single chain human
antibody, having a variable domain region from a heavy chain and a
variable domain region from a light chain and a peptide linker
connection the heavy chain and light chain variable domain regions,
wherein the heavy chain variable domain sequence that is at least
95% identical to the amino acid sequences selected from the group
consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO.
7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ
ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO.
25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, and combinations
thereof, and that has a light chain variable domain sequence that
is at least 95% identical to the amino acid sequences selected from
the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6,
SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID
NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24,
SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, and
combinations thereof. Preferably, the fully human single chain
antibody has both a heavy chain variable domain region and a light
chain variable domain region, wherein the single chain fully human
antibody has a heavy chain/light chain variable domain sequence
selected from the group consisting of SEQ ID NO. 1/SEQ ID NO. 2,
SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO.
7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID
NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16,
SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID
NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO.
25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID
NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, and combinations thereof.
[0026] The present disclosure further provides a method for
treating a broad spectrum of mammalian cancers or inflammatory
diseases or autoimmune diseases, comprising administering an
effective amount of an anti-IGF1R polypeptide, wherein the
anti-IGF1R polypeptide is selected from the group consisting of a
fully human antibody of an IgG class that binds to a IGF1R epitope
with a binding affinity of at least 10.sup.-6M, a fully human Fab
antibody fragment, having a variable domain region from a heavy
chain and a variable domain region from a light chain, a single
chain human antibody, having a variable domain region from a heavy
chain and a variable domain region from a light chain and a peptide
linker connection the heavy chain and light chain variable domain
regions, and combinations thereof;
[0027] wherein the fully human antibody has a heavy chain variable
domain sequence that is at least 95% identical to the amino acid
sequences selected from the group consisting of SEQ ID NO. 1, SEQ
ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11,
SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID
NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29,
SEQ ID NO. 31, and combinations thereof, and that has a light chain
variable domain sequence that is at least 95% identical to the
amino acid sequences selected from the group consisting of SEQ ID
NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ
ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO.
20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ
ID NO. 30, SEQ ID NO. 32, and combinations thereof;
[0028] wherein the fully human Fab antibody fragment has the heavy
chain variable domain sequence that is at least 95% identical to
the amino acid sequences selected from the group consisting of SEQ
ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9,
SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID
NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27,
SEQ ID NO. 29, SEQ ID NO. 31, and combinations thereof, and that
has the light chain variable domain sequence that is at least 95%
identical to the amino acid sequences selected from the group
consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO.
8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ
ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO.
26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, and combinations
thereof; and
[0029] wherein the single chain human antibody has the heavy chain
variable domain sequence that is at least 95% identical to the
amino acid sequences selected from the group consisting of SEQ ID
NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ
ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO.
19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ
ID NO. 29, SEQ ID NO. 31, and combinations thereof, and that has
the light chain variable domain sequence that is at least 95%
identical to the amino acid sequences selected from the group
consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO.
8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ
ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO.
26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, and combinations
thereof.
[0030] Preferably, the fully human antibody has both a heavy chain
and a light chain wherein the antibody has a heavy chain/light
chain variable domain sequence selected from the group consisting
of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO.
5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO.
10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ
ID NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO.
19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID
NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28,
SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, and
combinations thereof. Preferably, the fully human antibody Fab
fragment has both a heavy chain variable domain region and a light
chain variable domain region wherein the antibody has a heavy
chain/light chain variable domain sequence selected from the group
consisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4,
SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO.
9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID
NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18,
SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID
NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO.
27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID
NO. 32, and combinations thereof. Preferably, the fully human
single chain antibody has both a heavy chain variable domain region
and a light chain variable domain region, wherein the single chain
fully human antibody has a heavy chain/light chain variable domain
sequence selected from the group consisting of SEQ ID NO. 1/SEQ ID
NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID
NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ
ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO.
16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ
ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID NO. 24, SEQ ID NO.
25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID
NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, and combinations thereof.
[0031] Preferably, the broad spectrum of mammalian cancers to be
treated is selected from the group consisting of ovarian, colon,
breast, lung cancers, myelomas, neuroblastic-derived CNS tumors,
monocytic leukemias, B-cell derived leukemias, T-cell derived
leukemias, B-cell derived lymphomas, T-cell derived lymphomas, mast
cell derived tumors, and combinations thereof. Preferably, the
autoimmune disease or inflammatory disease is selected from the
group consisting of intestinal mucosal inflammation, wasting
disease associated with colitis, multiple sclerosis, systemic lupus
erythematosus, viral infections, rheumatoid arthritis,
osteoarthritis, psoriasis, Cohn's disease, and inflammatory bowel
disease.
[0032] An "antigen binding protein" is a protein comprising a
portion that binds to an antigen and, optionally, a scaffold or
framework portion that allows the antigen binding portion to adopt
a conformation that promotes binding of the antigen binding protein
to the antigen. Examples of antigen binding proteins include
antibodies, antibody fragments (e.g., an antigen binding portion of
an antibody), antibody derivatives, and antibody analogs. The
antigen binding protein can comprise, for example, an alternative
protein scaffold or artificial scaffold with grafted CDRs or CDR
derivatives. Such scaffolds include, but are not limited to,
antibody-derived scaffolds comprising mutations introduced to, for
example, stabilize the three-dimensional structure of the antigen
binding protein as well as wholly synthetic scaffolds comprising,
for example, a biocompatible polymer. See, for example, Korndorfer
et al., 2003, Proteins: Structure, Function, and Bioinformatics,
Volume 53, Issue 1:121-129; Roque et al., 2004, Biotechnol. Prog.
20:639-654. In addition, peptide antibody mimetics ("PAMs") can be
used, as well as scaffolds based on antibody mimetics utilizing
fibronection components as a scaffold.
[0033] An antigen binding protein can have, for example, the
structure of a naturally occurring immunoglobulin. An
"immunoglobulin" is a tetrameric molecule. In a naturally occurring
immunoglobulin, each tetramer is composed of two identical pairs of
polypeptide chains, each pair having one "light" (about 25 kDa) and
one "heavy" chain (about 50-70 kDa). The amino-terminal portion of
each chain includes a variable region of about 100 to 110 or more
amino acids primarily responsible for antigen recognition. The
carboxy-terminal portion of each chain defines a constant region
primarily responsible for effector function. Human light chains are
classified as kappa or lambda light chains. Heavy chains are
classified as mu, delta, gamma, alpha, or epsilon, and define the
antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
Within light and heavy chains, the variable and constant regions
are joined by a "J" region of about 12 or more amino acids, with
the heavy chain also including a "D" region of about 10 more amino
acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed.,
2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its
entirety for all purposes). The variable regions of each
light/heavy chain pair form the antibody binding site such that an
intact immunoglobulin has two binding sites.
[0034] The variable regions of naturally occurring immunoglobulin
chains exhibit the same general structure of relatively conserved
framework regions (FR) joined by three hypervariable regions, also
called complementarity determining regions or CDRs. From N-terminus
to C-terminus, both light and heavy chains comprise the domains
FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino
acids to each domain is in accordance with the definitions of Kabat
et al. in Sequences of Proteins of Immunological Interest, 5.sup.th
Ed., US Dept. of Health and Human Services, PHS, NIH, NIH
Publication no. 91-3242, 1991. Other numbering systems for the
amino acids in immunoglobulin chains include IMGT.RTM.
(international ImMunoGeneTics information system; Lefranc et al,
Dev. Comp. Immunol. 29:185-203; 2005) and AHo (Honegger and
Pluckthun, J. Mol. Biol. 309(3):657-670; 2001).
[0035] Antibodies can be obtained from sources such as serum or
plasma that contain immunoglobulins having varied antigenic
specificity. If such antibodies are subjected to affinity
purification, they can be enriched for a particular antigenic
specificity. Such enriched preparations of antibodies usually are
made of less than about 10% antibody having specific binding
activity for the particular antigen. Subjecting these preparations
to several rounds of affinity purification can increase the
proportion of antibody having specific binding activity for the
antigen. Antibodies prepared in this manner are often referred to
as "monospecific." Monospecfic antibody preparations can be made up
of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%,
95%, 97%, 99%, or 99.9% antibody having specific binding activity
for the particular antigen.
[0036] An "antibody" refers to an intact immunoglobulin or to an
antigen binding portion thereof that competes with the intact
antibody for specific binding, unless otherwise specified. Antigen
binding portions may be produced by recombinant DNA techniques or
by enzymatic or chemical cleavage of intact antibodies. Antigen
binding portions include, inter alia, Fab, Fab', F(ab').sub.2, Fv,
domain antibodies (dAbs), and complementarity determining region
(CDR) fragments, single-chain antibodies (scFv), chimeric
antibodies, diabodies, triabodies, tetrabodies, and polypeptides
that contain at least a portion of an immunoglobulin that is
sufficient to confer specific antigen binding to the
polypeptide.
[0037] A Fab fragment is a monovalent fragment having the V.sub.L,
V.sub.H, C.sub.L and C.sub.H1 domains; a F(ab').sub.2 fragment is a
bivalent fragment having two Fab fragments linked by a disulfide
bridge at the hinge region; a Fd fragment has the V.sub.H and
C.sub.H1 domains; an Fv fragment has the V.sub.L and V.sub.H
domains of a single arm of an antibody; and a dAb fragment has a
V.sub.H domain, a V.sub.L domain, or an antigen-binding fragment of
a V.sub.H or VL domain (U.S. Pat. Nos. 6,846,634; 6,696,245, US
App. Pub. 20/0202512; 2004/0202995; 2004/0038291; 2004/0009507;
2003/0039958, and Ward et al., Nature 341:544-546, 1989).
[0038] A single-chain antibody (scFv) is an antibody in which a
V.sub.L and a V.sub.H region are joined via a linker (e.g., a
synthetic sequence of amino acid residues) to form a continuous
protein chain wherein the linker is long enough to allow the
protein chain to fold back on itself and form a monovalent antigen
binding site (see, e.g., Bird et al., 1988, Science 242:423-26 and
Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83).
Diabodies are bivalent antibodies comprising two polypeptide
chains, wherein each polypeptide chain comprises V.sub.H and
V.sub.L domains joined by a linker that is too short to allow for
pairing between two domains on the same chain, thus allowing each
domain to pair with a complementary domain on another polypeptide
chain (see, e.g., Holliger et al., 1993, Proc. Natl. Acad. Sci. USA
90:6444-48, and Poljak et al., 1994, Structure 2:1121-23). If the
two polypeptide chains of a diabody are identical, then a diabody
resulting from their pairing will have two identical antigen
binding sites. Polypeptide chains having different sequences can be
used to make a diabody with two different antigen binding sites.
Similarly, tribodies and tetrabodies are antibodies comprising
three and four polypeptide chains, respectively, and forming three
and four antigen binding sites, respectively, which can be the same
or different.
[0039] Complementarity determining regions (CDRs) and framework
regions (FR) of a given antibody may be identified using the system
described by Kabat et al. supra; Lefranc et al., supra and/or
Honegger and Pluckthun, supra. One or more CDRs may be incorporated
into a molecule either covalently or noncovalently to make it an
antigen binding protein. An antigen binding protein may incorporate
the CDR(s) as part of a larger polypeptide chain, may covalently
link the CDR(s) to another polypeptide chain, or may incorporate
the CDR(s) noncovalently. The CDRs permit the antigen binding
protein to specifically bind to a particular antigen of
interest.
[0040] An antigen binding protein may have one or more binding
sites. If there is more than one binding site, the binding sites
may be identical to one another or may be different. For example, a
naturally occurring human immunoglobulin typically has two
identical binding sites, while a "bispecific" or "bifunctional"
antibody has two different binding sites.
[0041] The term "human antibody" includes all antibodies that have
one or more variable and constant regions derived from human
immunoglobulin sequences. In one embodiment, all of the variable
and constant domains are derived from human immunoglobulin
sequences (a fully human antibody). These antibodies may be
prepared in a variety of ways, examples of which are described
below, including through the immunization with an antigen of
interest of a mouse that is genetically modified to express
antibodies derived from human heavy and/or light chain-encoding
genes.
[0042] A humanized antibody has a sequence that differs from the
sequence of an antibody derived from a non-human species by one or
more amino acid substitutions, deletions, and/or additions, such
that the humanized antibody is less likely to induce an immune
response, and/or induces a less severe immune response, as compared
to the non-human species antibody, when it is administered to a
human subject. In one embodiment, certain amino acids in the
framework and constant domains of the heavy and/or light chains of
the non-human species antibody are mutated to produce the humanized
antibody. In another embodiment, the constant domain(s) from a
human antibody are fused to the variable domain(s) of a non-human
species. In another embodiment, one or more amino acid residues in
one or more CDR sequences of a non-human antibody are changed to
reduce the likely immunogenicity of the non-human antibody when it
is administered to a human subject, wherein the changed amino acid
residues either are not critical for immunospecific binding of the
antibody to its antigen, or the changes to the amino acid sequence
that are made are conservative changes, such that the binding of
the humanized antibody to the antigen is not significantly worse
than the binding of the non-human antibody to the antigen. Examples
of how to make humanized antibodies may be found in U.S. Pat. Nos.
6,054,297, 5,886,152 and 5,877,293.
[0043] The term "chimeric antibody" refers to an antibody that
contains one or more regions from one antibody and one or more
regions from one or more other antibodies. In one embodiment, one
or more of the CDRs are derived from a human anti-IGF1R antibody.
In another embodiment, all of the CDRs are derived from a human
anti-IGF1R antibody. In another embodiment, the CDRs from more than
one human anti-IGF1R antibodies are mixed and matched in a chimeric
antibody. For instance, a chimeric antibody may comprise a CDR1
from the light chain of a first human anti-PAR-2 antibody, a CDR2
and a CDR3 from the light chain of a second human anti-IGF1R
antibody, and the CDRs from the heavy chain from a third anti-IGF1R
antibody. Other combinations are possible.
[0044] Further, the framework regions may be derived from one of
the same anti-IGF1R antibodies, from one or more different
antibodies, such as a human antibody, or from a humanized antibody.
In one example of a chimeric antibody, a portion of the heavy
and/or light chain is identical with, homologous to, or derived
from an antibody from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is/are identical with, homologous to, or derived from an
antibody (-ies) from another species or belonging to another
antibody class or subclass. Also included are fragments of such
antibodies that exhibit the desired biological activity (i.e., the
ability to specifically bind IGF1R).
[0045] A "neutralizing antibody" or an "inhibitory antibody" is an
antibody that inhibits the proteolytic activation of IGF1R when an
excess of the anti-IGF1R antibody reduces the amount of activation
by at least about 20% using an assay such as those described herein
in the Examples. In various embodiments, the antigen binding
protein reduces the amount of amount of proteolytic activation of
IGF1R by at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,
97%, 99%, and 99.9%.
[0046] Fragments or analogs of antibodies can be readily prepared
by those of ordinary skill in the art following the teachings of
this specification and using techniques known in the art. Preferred
amino- and carboxy-termini of fragments or analogs occur near
boundaries of functional domains. Structural and functional domains
can be identified by comparison of the nucleotide and/or amino acid
sequence data to public or proprietary sequence databases.
Computerized comparison methods can be used to identify sequence
motifs or predicted protein conformation domains that occur in
other proteins of known structure and/or function. Methods to
identify protein sequences that fold into a known three-dimensional
structure are known. See, Bowie et al., 1991, Science 253:164.
[0047] A "CDR grafted antibody" is an antibody comprising one or
more CDRs derived from an antibody of a particular species or
isotype and the framework of another antibody of the same or
different species or isotype.
[0048] A "multi-specific antibody" is an antibody that recognizes
more than one epitope on one or more antigens. A subclass of this
type of antibody is a "bi-specific antibody" which recognizes two
distinct epitopes on the same or different antigens.
[0049] An antigen binding protein "specifically binds" to an
antigen (e.g., human IGF1R) if it binds to the antigen with a
dissociation constant of 1 nanomolar or less.
[0050] An "antigen binding domain," "antigen binding region," or
"antigen binding site" is a portion of an antigen binding protein
that contains amino acid residues (or other moieties) that interact
with an antigen and contribute to the antigen binding protein's
specificity and affinity for the antigen. For an antibody that
specifically binds to its antigen, this will include at least part
of at least one of its CDR domains.
[0051] An "epitope" is the portion of a molecule that is bound by
an antigen binding protein (e.g., by an antibody). An epitope can
comprise non-contiguous portions of the molecule (e.g., in a
polypeptide, amino acid residues that are not contiguous in the
polypeptide's primary sequence but that, in the context of the
polypeptide's tertiary and quaternary structure, are near enough to
each other to be bound by an antigen binding protein).
[0052] The "percent identity" of two polynucleotide or two
polypeptide sequences is determined by comparing the sequences
using the GAP computer program (a part of the GCG Wisconsin
Package, version 10.3 (Accelrys, San Diego, Calif.)) using its
default parameters.
[0053] The terms "polynucleotide," "oligonucleotide" and "nucleic
acid" are used interchangeably throughout and include DNA molecules
(e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of
the DNA or RNA generated using nucleotide analogs (e.g., peptide
nucleic acids and non-naturally occurring nucleotide analogs), and
hybrids thereof. The nucleic acid molecule can be single-stranded
or double-stranded. In one embodiment, the nucleic acid molecules
of the invention comprise a contiguous open reading frame encoding
an antibody, or a fragment, derivative, mutein, or variant
thereof.
[0054] Two single-stranded polynucleotides are "the complement" of
each other if their sequences can be aligned in an anti-parallel
orientation such that every nucleotide in one polynucleotide is
opposite its complementary nucleotide in the other polynucleotide,
without the introduction of gaps, and without unpaired nucleotides
at the 5' or the 3' end of either sequence. A polynucleotide is
"complementary" to another polynucleotide if the two
polynucleotides can hybridize to one another under moderately
stringent conditions. Thus, a polynucleotide can be complementary
to another polynucleotide without being its complement.
[0055] A "vector" is a nucleic acid that can be used to introduce
another nucleic acid linked to it into a cell. One type of vector
is a "plasmid," which refers to a linear or circular double
stranded DNA molecule into which additional nucleic acid segments
can be ligated. Another type of vector is a viral vector (e.g.,
replication defective retroviruses, adenoviruses and
adeno-associated viruses), wherein additional DNA segments can be
introduced into the viral genome. Certain vectors are capable of
autonomous replication in a host cell into which they are
introduced (e.g., bacterial vectors comprising a bacterial origin
of replication and episomal mammalian vectors). Other vectors
(e.g., non-episomal mammalian vectors) are integrated into the
genome of a host cell upon introduction into the host cell, and
thereby are replicated along with the host genome. An "expression
vector" is a type of vector that can direct the expression of a
chosen polynucleotide.
[0056] A nucleotide sequence is "operably linked" to a regulatory
sequence if the regulatory sequence affects the expression (e.g.,
the level, timing, or location of expression) of the nucleotide
sequence. A "regulatory sequence" is a nucleic acid that affects
the expression (e.g., the level, timing, or location of expression)
of a nucleic acid to which it is operably linked. The regulatory
sequence can, for example, exert its effects directly on the
regulated nucleic acid, or through the action of one or more other
molecules (e.g., polypeptides that bind to the regulatory sequence
and/or the nucleic acid). Examples of regulatory sequences include
promoters, enhancers and other expression control elements (e.g.,
polyadenylation signals).
[0057] A "host cell" is a cell that can be used to express a
nucleic acid, e.g., a nucleic acid of the invention. A host cell
can be a prokaryote, for example, E. coli, or it can be a
eukaryote, for example, a single-celled eukaryote (e.g., a yeast or
other fungus), a plant cell (e.g., a tobacco or tomato plant cell),
an animal cell (e.g., a human cell, a monkey cell, a hamster cell,
a rat cell, a mouse cell, or an insect cell) or a hybridoma.
Examples of host cells include the COS-7 line of monkey kidney
cells (ATCC CRL 1651), L cells, C127 cells, 3T3 cells (ATCC CCL
163), Chinese hamster ovary (CHO) cells or their derivatives such
as Veggie CHO and related cell lines which grow in serum-free media
(see Rasmussen et al., 1998, Cytotechnology 28:31) or CHO strain
DX-B 11, which is deficient in DHFR (see Urlaub et al., 1980, Proc.
Natl. Acad. Sci. USA 77:4216-20), HeLa cells, BHK (ATCC CRL 10)
cell lines, the CV1/EBNA cell line derived from the African green
monkey kidney cell line CV1 (ATCC CCL 70) (see McMahan et al.,
1991, EMBO J. 10:2821), human embryonic kidney cells such as
293,293 EBNA or MSR 293, human epidermal A431 cells, human Colo205
cells, other transformed primate cell lines, normal diploid cells,
cell strains derived from in vitro culture of primary tissue,
primary explants, HL-60, U937, HaK or Jurkat cells. Typically, a
host cell is a cultured cell that can be transformed or transfected
with a polypeptide-encoding nucleic acid, which can then be
expressed in the host cell. The phrase "recombinant host cell" can
be used to denote a host cell that has been transformed or
transfected with a nucleic acid to be expressed. A host cell also
can be a cell that comprises the nucleic acid but does not express
it at a desired level unless a regulatory sequence is introduced
into the host cell such that it becomes operably linked with the
nucleic acid. It is understood that the term host cell refers not
only to the particular subject cell but also to the progeny or
potential progeny of such a cell. Because certain modifications may
occur in succeeding generations due to, e.g., mutation or
environmental influence, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein.
[0058] Preferably, the mammalian cancer to be treated is selected
from the group consisting of the osteosarcoma, rhabdomyosarcoma,
neuroblastoma, any pediatric cancer, kidney cancer, leukemia, renal
transitional cell cancer, Werner-Morrison syndrome, bladder cancer,
Wilm's cancer, ovarian cancer, pancreatic cancer, benign prostatic
hyperplasia, breast cancer, prostate cancer, bone cancer, lung
cancer, gastric cancer, colorectal cancer, cervical cancer,
synovial sarcoma, diarrhea associated with metastatic carcinoid,
vasoactive intestinal peptide secreting tumors, head and neck
cancer, squamous cell carcinoma, multiple myeloma, solitary
plasmacytoma, renal cell cancer, retinoblastoma, germ cell tumors,
hepatoblastoma, hepatocellular carcinoma, melanoma, rhabdoid tumor
of the kidney, Ewing Sarcoma, chondrosarcoma, haemotological
malignancy, chronic lymphoblastic leukemia, chronic myelomonocytic
leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia,
acute myelogenous leukemia, acute myeloblastic leukemia, chronic
myeloblastic leukemia, Hodgkin's disease, non-Hodgkin's lymphoma,
chronic lymphocytic leukemia, chronic myelogenous leukemia,
myelodysplastic syndrome, hairy cell leukemia, mast cell leukemia,
mast cell neoplasm, follicular lymphoma, diffuse large cell
lymphoma, mantle cell lymphoma, Burkitt Lymphoma, mycosis
fungoides, seary syndrome, cutaneous T-cell lymphoma, chronic
myeloproliferative disorders, a central nervous system tumor, brain
cancer, glioblastoma, non-glioblastoma brain cancer, meningioma,
pituitary adenoma, vestibular schwannoma, a primitive
neuroectodermal tumor, medulloblastoma, astrocytoma, anaplastic
astrocytoma, oligodendroglioma, ependymoma and choroid plexus
papilloma, a myeloproliferative disorder, polycythemia vera,
thrombocythemia, idiopathic myelfibrosis, soft tissue sarcoma,
thyroid cancer, endometrial cancer, carcinoid cancer, germ cell
tumors, liver cancer, and combinations thereof.
[0059] The expression construct is introduced into the host cell
using a method appropriate to the host cell. A variety of methods
for introducing nucleic acids into host cells are known in the art,
including, but not limited to, electroporation; transfection
employing calcium chloride, rubidium chloride, calcium phosphate,
DEAE-dextran, or other substances; microprojectile bombardment;
lipofection; and infection (where the vector is an infectious
agent). Suitable host cells include prokaryotes, yeast, mammalian
cells, or bacterial cells.
[0060] Suitable bacteria include gram negative or gram positive
organisms, for example, E. coli or Bacillus spp. Yeast, preferably
from the Saccharomyces species, such as S. cerevisiae, may also be
used for production of polypeptides. Various mammalian or insect
cell culture systems can also be employed to express recombinant
proteins. Baculovirus systems for production of heterologous
proteins in insect cells are reviewed by Luckow and Summers,
(Bio/Technology, 6:47, 1988). Examples of suitable mammalian host
cell lines include endothelial cells, COS-7 monkey kidney cells,
CV-1, L cells, C127, 3T3, Chinese hamster ovary (CHO), human
embryonic kidney cells, HeLa, 293, 293T, and BHK cell lines.
Purified polypeptides are prepared by culturing suitable
host/vector systems to express the recombinant proteins. For many
applications, the small size of many of the polypeptides disclosed
herein would make expression in E. coli as the preferred method for
expression. The protein is then purified from culture media or cell
extracts.
[0061] Proteins disclosed herein can also be produced using
cell-translation systems. For such purposes the nucleic acids
encoding the polypeptide must be modified to allow in vitro
transcription to produce mRNA and to allow cell-free translation of
the mRNA in the particular cell-free system being utilized
(eukaryotic such as a mammalian or yeast cell-free translation
system or prokaryotic such as a bacterial cell-free translation
system.
[0062] IGF1R-binding polypeptides can also be produced by chemical
synthesis (e.g., by the methods described in Solid Phase Peptide
Synthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, Ill.).
Modifications to the protein can also be produced by chemical
synthesis.
[0063] The polypeptides of the present disclosure can be purified
by isolation/purification methods for proteins generally known in
the field of protein chemistry. Non-limiting examples include
extraction, recrystallization, salting out (e.g., with ammonium
sulfate or sodium sulfate), centrifugation, dialysis,
ultrafiltration, adsorption chromatography, ion exchange
chromatography, hydrophobic chromatography, normal phase
chromatography, reversed-phase chromatography, gel filtration, gel
permeation chromatography, affinity chromatography,
electrophoresis, countercurrent distribution or any combinations of
these. After purification, polypeptides may be exchanged into
different buffers and/or concentrated by any of a variety of
methods known to the art, including, but not limited to, filtration
and dialysis.
[0064] The purified polypeptide is preferably at least 85% pure,
more preferably at least 95% pure, and most preferably at least 98%
pure. Regardless of the exact numerical value of the purity, the
polypeptide is sufficiently pure for use as a pharmaceutical
product.
Post-Translational Modifications of Polypeptides
[0065] In certain embodiments, the binding polypeptides of the
invention may further comprise post-translational modifications.
Exemplary post-translational protein modifications include
phosphorylation, acetylation, methylation, ADP-ribosylation,
ubiquitination, glycosylation, carbonylation, sumoylation,
biotinylation or addition of a polypeptide side chain or of a
hydrophobic group. As a result, the modified soluble polypeptides
may contain non-amino acid elements, such as lipids, poly- or
mono-saccharide, and phosphates. A preferred form of glycosylation
is sialylation, which conjugates one or more sialic acid moieties
to the polypeptide. Sialic acid moieties improve solubility and
serum half-life while also reducing the possible immunogeneticity
of the protein. See Raju et al. Biochemistry. 2001 31;
40(30):8868-76.
[0066] In one specific embodiment, modified forms of the subject
soluble polypeptides comprise linking the subject soluble
polypeptides to nonproteinaceous polymers. In one specific
embodiment, the polymer is polyethylene glycol ("PEG"),
polypropylene glycol, or polyoxyalkylenes, in the manner as set
forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417;
4,791,192 or 4,179,337.
[0067] PEG is a water soluble polymer that is commercially
available or can be prepared by ring-opening polymerization of
ethylene glycol according to methods well known in the art (Sandler
and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3,
pages 138-161). The term "PEG" is used broadly to encompass any
polyethylene glycol molecule, without regard to size or to
modification at an end of the PEG, and can be represented by the
formula: X--O(CH.sub.2CH.sub.2O).sub.n-1CH.sub.2CH.sub.2OH (1),
where n is 20 to 2300 and X is H or a terminal modification, e.g.,
a C.sub.1-4 alkyl. In one embodiment, the PEG of the invention
terminates on one end with hydroxy or methoxy, i.e., X is H or
CH.sub.3 ("methoxy PEG"). A PEG can contain further chemical groups
which are necessary for binding reactions; which results from the
chemical synthesis of the molecule; or which is a spacer for
optimal distance of parts of the molecule. In addition, such a PEG
can consist of one or more PEG side-chains which are linked
together. PEGs with more than one PEG chain are called multiarmed
or branched PEGs. Branched PEGs can be prepared, for example, by
the addition of polyethylene oxide to various polyols, including
glycerol, pentaerythriol, and sorbitol. For example, a four-armed
branched PEG can be prepared from pentaerythriol and ethylene
oxide. Branched PEG are described in, for example, EP-A 0 473 084
and U.S. Pat. No. 5,932,462. One form of PEGs includes two PEG
side-chains (PEG2) linked via the primary amino groups of a lysine
(Monfardini et al., Bioconjugate Chem. 6 (1995) 62-69).
[0068] In a preferred embodiment, the pegylated.sup.10Fn3
polypeptide is produced by site-directed pegylation, particularly
by conjugation of PEG to a cysteine moiety at the N- or C-terminus.
Accordingly, the present disclosure provides a target-binding
.sup.10Fn3 polypeptide with improved pharmacokinetic properties,
the polypeptide comprising: a .sup.10Fn3 domain having from about
80 to about 150 amino acids, wherein at least one of the loops of
said .sup.10Fn3 domain participate in target binding; and a
covalently bound PEG moiety, wherein said .sup.10Fn3 polypeptide
binds to the target with a K.sub.D of less than 100 nM and has a
clearance rate of less than 30 mL/hr/kg in a mammal. The PEG moiety
may be attached to the .sup.10Fn3 polypeptide by site directed
pegylation, such as by attachment to a Cys residue, where the Cys
residue may be positioned at the N-terminus of the .sup.0Fn3
polypeptide or between the N-terminus and the most N-terminal beta
or beta-like strand or at the C-terminus of the .sup.10Fn3
polypeptide or between the C-terminus and the most C-terminal beta
or beta-like strand. A Cys residue may be situated at other
positions as well, particularly any of the loops that do not
participate in target binding. A PEG moiety may also be attached by
other chemistry, including by conjugation to amines.
[0069] PEG conjugation to peptides or proteins generally involves
the activation of PEG and coupling of the activated
PEG-intermediates directly to target proteins/peptides or to a
linker, which is subsequently activated and coupled to target
proteins/peptides (see Abuchowski et al., J. Biol. Chem., 252, 3571
(1977) and J. Biol. Chem., 252, 3582 (1977), Zalipsky, et al., and
Harris et. al., in: Poly(ethylene glycol) Chemistry: Biotechnical
and Biomedical Applications; (Harris ed.) Plenum Press: New York,
1992; Chap. 21 and 22). It is noted that a binding polypeptide
containing a PEG molecule is also known as a conjugated protein,
whereas the protein lacking an attached PEG molecule can be
referred to as unconjugated.
[0070] A variety of molecular mass forms of PEG can be selected,
e.g., from about 1,000 Daltons (Da) to 100,000 Da (n is 20 to
2300), for conjugating to IGF1R-binding polypeptides. The number of
repeating units "n" in the PEG is approximated for the molecular
mass described in Daltons. It is preferred that the combined
molecular mass of PEG on an activated linker is suitable for
pharmaceutical use. Thus, in one embodiment, the molecular mass of
the PEG molecules does not exceed 100,000 Da. For example, if three
PEG molecules are attached to a linker, where each PEG molecule has
the same molecular mass of 12,000 Da (each n is about 270), then
the total molecular mass of PEG on the linker is about 36,000 Da
(total n is about 820). The molecular masses of the PEG attached to
the linker can also be different, e.g., of three molecules on a
linker two PEG molecules can be 5,000 Da each (each n is about 110)
and one PEG molecule can be 12,000 Da (n is about 270).
[0071] In a specific embodiment of the disclosure an IGF1R binding
polypeptide is covalently linked to one poly(ethylene glycol) group
of the formula:
--CO--(CH.sub.2).sub.x--(OCH.sub.2CH.sub.2).sub.m--OR, with the
--CO (i.e. carbonyl) of the poly(ethylene glycol) group forming an
amide bond with one of the amino groups of the binding polypeptide;
R being lower alkyl; x being 2 or 3; m being from about 450 to
about 950; and n and m being chosen so that the molecular weight of
the conjugate minus the binding polypeptide is from about 10 to 40
kDa. In one embodiment, a binding polypeptide's 6-amino group of a
lysine is the available (free) amino group.
[0072] The above conjugates may be more specifically presented by
formula (II):
P--NHCO--(CH.sub.2).sub.x--(OCH.sub.2CH.sub.2).sub.m--OR (II),
wherein P is the group of a binding polypeptide as described
herein, (i.e. without the amino group or amino groups which form an
amide linkage with the carbonyl shown in formula (II); and wherein
R is lower alkyl; x is 2 or 3; m is from about 450 to about 950 and
is chosen so that the molecular weight of the conjugate minus the
binding polypeptide is from about 10 to about 40 kDa. As used
herein, the given ranges of "m" have an orientational meaning. The
ranges of "m" are determined in any case, and exactly, by the
molecular weight of the PEG group.
[0073] One skilled in the art can select a suitable molecular mass
for PEG, e.g., based on how the pegylated binding polypeptide will
be used therapeutically, the desired dosage, circulation time,
resistance to proteolysis, immunogenicity, and other
considerations.
[0074] In one specific embodiment, carbonate esters of PEG are used
to form the PEG-binding polypeptide conjugates.
N,N'-disuccinimidylcarbonate (DSC) may be used in the reaction with
PEG to form active mixed PEG-succinimidyl carbonate that may be
subsequently reacted with a nucleophilic group of a linker or an
amino group of a binding polypeptide (see U.S. Pat. Nos. 5,281,698
and 5,932,462). In a similar type of reaction,
1,1'-(dibenzotriazolyl)carbonate and di-(2-pyridyl)carbonate may be
reacted with PEG to form PEG-benzotriazolyl and PEG-pyridyl mixed
carbonate (U.S. Pat. No. 5,382,657), respectively.
[0075] Pegylation of a .sup.10Fn3 polypeptide can be performed
according to the methods of the state of the art, for example by
reaction of the binding polypeptide with electrophilically active
PEGs (supplier: Shearwater Corp., USA, www.shearwatercorp.com).
Preferred PEG reagents of the present invention are, e.g.,
N-hydroxysuccinimidyl propionates (PEG-SPA), butanoates (PEG-SBA),
PEG-succinimidyl propionate or branched N-hydroxysuccinimides such
as mPEG2-NHS (Monfardini et al., Bioconjugate Chem. 6 (1995)
62-69). Such methods may used to pegylated at an f-amino group of a
binding polypeptide lysine or the N-terminal amino group of the
binding polypeptide.
[0076] In another embodiment, PEG molecules may be coupled to
sulfhydryl groups on a binding polypeptide (Sartore et al., Appl.
Biochem. Biotechnol., 27, 45 (1991); Morpurgo et al., Biocon.
Chem., 7, 363-368 (1996); Goodson et al., Bio/Technology (1990) 8,
343; U.S. Pat. No. 5,766,897). U.S. Pat. Nos. 6,610,281 and
5,766,897 describes exemplary reactive PEG species that may be
coupled to sulfhydryl groups.
[0077] In some embodiments where PEG molecules are conjugated to
cysteine residues on a binding polypeptide, the cysteine residues
are native to the binding polypeptide, whereas in other
embodiments, one or more cysteine residues are engineered into the
binding polypeptide. Mutations may be introduced into a binding
polypeptide coding sequence to generate cysteine residues. This
might be achieved, for example, by mutating one or more amino acid
residues to cysteine. Preferred amino acids for mutating to a
cysteine residue include serine, threonine, alanine and other
hydrophilic residues. Preferably, the residue to be mutated to
cysteine is a surface-exposed residue. Alternatively, surface
residues may be predicted by comparing the amino acid sequences of
binding polypeptides, given that the crystal structure of the
framework based on which binding polypeptides are designed and
evolved has been solved (Himanen et al., Nature. (2001) 20-27;
414(6866):933-8) and thus the surface-exposed residues identified.
In one embodiment, cysteine residues are introduced into binding
polypeptides at or near the N- and/or C-terminus, or within loop
regions.
[0078] In some embodiments, the pegylated binding polypeptide
comprises a PEG molecule covalently attached to the alpha amino
group of the N-terminal amino acid. Site specific N-terminal
reductive amination is described in Pepinsky et al., (2001) JPET,
297, 1059, and U.S. Pat. No. 5,824,784. The use of a PEG-aldehyde
for the reductive amination of a protein utilizing other available
nucleophilic amino groups is described in U.S. Pat. No. 4,002,531,
in Wieder et al., (1979) J. Biol. Chem. 254, 12579, and in Chamow
et al., (1994) Bioconjugate Chem. 5, 133.
[0079] In another embodiment, pegylated binding polypeptide
comprises one or more PEG molecules covalently attached to a
linker, which in turn is attached to the alpha amino group of the
amino acid residue at the N-terminus of the binding polypeptide.
Such an approach is disclosed in U.S. Patent Publication
2002/0044921 and in WO094/01451.
[0080] In one embodiment, a binding polypeptide is pegylated at the
C-terminus. In a specific embodiment, a protein is pegylated at the
C-terminus by the introduction of C-terminal azido-methionine and
the subsequent conjugation of a methyl-PEG-triarylphosphine
compound via the Staudinger reaction. This C-terminal conjugation
method is described in Cazalis et al., Bioconjug. Chem. 2004;
15(5):1005-1009.
[0081] Monopegylation of a binding polypeptide can also be produced
according to the general methods described in WO 94/01451. WO
94/01451 describes a method for preparing a recombinant polypeptide
with a modified terminal amino acid alpha-carbon reactive group.
The steps of the method involve forming the recombinant polypeptide
and protecting it with one or more biologically added protecting
groups at the N-terminal alpha-amine and C-terminal alpha-carboxyl.
The polypeptide can then be reacted with chemical protecting agents
to selectively protect reactive side chain groups and thereby
prevent side chain groups from being modified. The polypeptide is
then cleaved with a cleavage reagent specific for the biological
protecting group to form an unprotected terminal amino acid
alpha-carbon reactive group. The unprotected terminal amino acid
alpha-carbon reactive group is modified with a chemical modifying
agent. The side chain protected terminally modified single copy
polypeptide is then deprotected at the side chain groups to form a
terminally modified recombinant single copy polypeptide. The number
and sequence of steps in the method can be varied to achieve
selective modification at the N- and/or C-terminal amino acid of
the polypeptide.
[0082] The ratio of a binding polypeptide to activated PEG in the
conjugation reaction can be from about 1:0.5 to 1:50, between from
about 1:1 to 1:30, or from about 1:5 to 1:15. Various aqueous
buffers can be used in the present method to catalyze the covalent
addition of PEG to the binding polypeptide. In one embodiment, the
pH of a buffer used is from about 7.0 to 9.0. In another
embodiment, the pH is in a slightly basic range, e.g., from about
7.5 to 8.5. Buffers having a pKa close to neutral pH range may be
used, e.g., phosphate buffer.
[0083] Conventional separation and purification techniques known in
the art can be used to purify PEGylated binding polypeptide, such
as size exclusion (e.g. gel filtration) and ion exchange
chromatography. Products may also be separated using SDS-PAGE.
Products that may be separated include mono-, di-, tri- poly- and
un-pegylated binding polypeptide, as well as free PEG. The
percentage of mono-PEG conjugates can be controlled by pooling
broader fractions around the elution peak to increase the
percentage of mono-PEG in the composition. About ninety percent
mono-PEG conjugates represents a good balance of yield and
activity. Compositions in which, for example, at least ninety-two
percent or at least ninety-six percent of the conjugates are
mono-PEG species may be desired. In an embodiment of this invention
the percentage of mono-PEG conjugates is from ninety percent to
ninety-six percent.
[0084] In one embodiment, PEGylated binding polypeptide of the
invention contain one, two or more PEG moieties. In one embodiment,
the PEG moiety(ies) are bound to an amino acid residue which is on
the surface of the protein and/or away from the surface that
contacts the target ligand. In one embodiment, the combined or
total molecular mass of PEG in PEG-binding polypeptide is from
about 3,000 Da to 60,000 Da, optionally from about 10,000 Da to
36,000 Da. In a one embodiment, the PEG in pegylated binding
polypeptide is a substantially linear, straight-chain PEG.
[0085] In one embodiment of the invention, the PEG in pegylated
binding polypeptide is not hydrolyzed from the pegylated amino acid
residue using a hydroxylamine assay, e.g., 450 mM hydroxylamine (pH
6.5) over 8 to 16 hours at room temperature, and is thus stable. In
one embodiment, greater than 80% of the composition is stable
mono-PEG-binding polypeptide, more preferably at least 90%, and
most preferably at least 95%.
[0086] In another embodiment, the pegylated binding polypeptides of
the invention will preferably retain at least 25%, 50%, 60%, 70%,
80%, 85%, 90%, 95% or 100% of the biological activity associated
with the unmodified protein. In one embodiment, biological activity
refers to its ability to bind to IGF1R, as assessed by KD, k.sub.on
or k.sub.off. In one specific embodiment, the pegylated binding
polypeptide protein shows an increase in binding to IGF1R relative
to unpegylated binding polypeptide.
[0087] The serum clearance rate of PEG-modified polypeptide may be
decreased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or even
90%, relative to the clearance rate of the unmodified binding
polypeptide. The PEG-modified polypeptide may have a half-life
(t.sub.1/2) which is enhanced relative to the half-life of the
unmodified protein. The half-life of PEG-binding polypeptide may be
enhanced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
100%, 125%, 150%, 175%, 200%, 250%, 300%, 400% or 500%, or even by
1000% relative to the half-life of the unmodified binding
polypeptide. In some embodiments, the protein half-life is
determined in vitro, such as in a buffered saline solution or in
serum. In other embodiments, the protein half-life is an in vivo
half life, such as the half-life of the protein in the serum or
other bodily fluid of an animal.
Therapeutic Formulations and Modes of Administration
[0088] The present disclosure features methods for treating
conditions or preventing pre-conditions which respond to an
inhibition of IGF1R biological activity. Preferred examples are
conditions that are characterized by inflammation or cellular
hyperproliferation. Techniques and dosages for administration vary
depending on the type of specific polypeptide and the specific
condition being treated but can be readily determined by the
skilled artisan. In general, regulatory agencies require that a
protein reagent to be used as a therapeutic is formulated so as to
have acceptably low levels of pyrogens. Accordingly, therapeutic
formulations will generally be distinguished from other
formulations in that they are substantially pyrogen free, or at
least contain no more than acceptable levels of pyrogen as
determined by the appropriate regulatory agency (e.g., FDA).
[0089] Therapeutic compositions of the present disclosure may be
administered with a pharmaceutically acceptable diluent, carrier,
or excipient, in unit dosage form. Administration may be parenteral
(e.g., intravenous, subcutaneous), oral, or topical, as
non-limiting examples. In addition, any gene therapy technique,
using nucleic acids encoding the polypeptides of the invention, may
be employed, such as naked DNA delivery, recombinant genes and
vectors, cell-based delivery, including ex vivo manipulation of
patients' cells, and the like.
[0090] The composition can be in the form of a pill, tablet,
capsule, liquid, or sustained release tablet for oral
administration; or a liquid for intravenous, subcutaneous or
parenteral administration; gel, lotion, ointment, cream, or a
polymer or other sustained release vehicle for local
administration.
[0091] Methods well known in the art for making formulations are
found, for example, in "Remington: The Science and Practice of
Pharmacy" (20th ed., ed. A. R. Gennaro A R., 2000, Lippincott
Williams & Wilkins, Philadelphia, Pa.). Formulations for
parenteral administration may, for example, contain excipients,
sterile water, saline, polyalkylene glycols such as polyethylene
glycol, oils of vegetable origin, or hydrogenated napthalenes.
Biocompatible, biodegradable lactide polymer, lactide/glycolide
copolymer, or polyoxyethylene-polyoxypropylene copolymers may be
used to control the release of the compounds. Nanoparticulate
formulations (e.g., biodegradable nanoparticles, solid lipid
nanoparticles, liposomes) may be used to control the
biodistribution of the compounds. Other potentially useful
parenteral delivery systems include ethylene-vinyl acetate
copolymer particles, osmotic pumps, implantable infusion systems,
and liposomes. The concentration of the compound in the formulation
varies depending upon a number of factors, including the dosage of
the drug to be administered, and the route of administration.
[0092] The polypeptide may be optionally administered as a
pharmaceutically acceptable salt, such as non-toxic acid addition
salts or metal complexes that are commonly used in the
pharmaceutical industry. Examples of acid addition salts include
organic acids such as acetic, lactic, pamoic, maleic, citric,
malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic,
tartaric, methanesulfonic, toluenesulfonic, or trifluoroacetic
acids or the like; polymeric acids such as tannic acid,
carboxymethyl cellulose, or the like; and inorganic acid such as
hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid,
or the like. Metal complexes include zinc, iron, and the like. In
one example, the polypeptide is formulated in the presence of
sodium acetate to increase thermal stability.
[0093] A therapeutically effective dose refers to a dose that
produces the therapeutic effects for which it is administered. The
exact dose will depend on the disorder to be treated, and may be
ascertained by one skilled in the art using known techniques. In
general, the polypeptide is administered at about 0.01 .mu.g/kg to
about 50 mg/kg per day, preferably 0.01 mg/kg to about 30 mg/kg per
day, most preferably 0.1 mg/kg to about 20 mg/kg per day. The
polypeptide may be given daily (e.g., once, twice, three times, or
four times daily) or preferably less frequently (e.g., weekly,
every two weeks, every three weeks, monthly, or quarterly). In
addition, as is known in the art, adjustments for age as well as
the body weight, general health, sex, diet, time of administration,
drug interaction, and the severity of the disease may be necessary,
and will be ascertainable with routine experimentation by those
skilled in the art.
Exemplary Uses
[0094] The IGF1R binding proteins described herein and their
related variants are useful in a number of therapeutic and
diagnostic applications. These include the inhibition of the
biological activity of IGF1R by competing for or blocking the
binding to an IGF1R as well as the delivery of cytotoxic or imaging
moieties to cells, preferably cells expressing IGF1R.
[0095] On the basis of their efficacy as inhibitors of IGF1R
biological activity, the polypeptides of this disclosure are
effective against a number of cancer conditions as well as
complications arising from cancer, such as pleural effusion and
ascites. Preferably, the IGF1R-binding polypeptides of the
disclosure can be used for the treatment of prevention of
hyperproliferative diseases or cancer and the metastatic spread of
cancers. Preferred indications for the disclosed
anti-IGF1Rantibodies include colorectal cancers, head and neck
cancers, small cell lung cancer, non-small cell lung cancer (NSCLC)
and pancreatic cancer. Non-limiting examples of cancers include
bladder, blood, bone, brain, breast, cartilage, colon kidney,
liver, lung, lymph node, nervous tissue, ovary, pancreatic,
prostate, skeletal muscle, skin, spinal cord, spleen, stomach,
testes, thymus, thyroid, trachea, urogenital tract, ureter,
urethra, uterus, or vaginal cancer.
[0096] In addition, various inflammatory disorders can be treated
with the disclosed anti-IGF1R binding polypeptides disclosed
herein. Such inflammatory disorders include, for example,
intestinal mucosa inflammation wasting diseases associated with
colitis, multiple sclerosis, systemic lupus erythematosus, viral
infections, rheumatoid arthritis, osteoarthritis, psoriasis, and
Crohn's disease.
[0097] A IGF1R binding polypeptide can be administered alone or in
combination with one or more additional therapies such as
chemotherapy radiotherapy, immunotherapy, surgical intervention, or
any combination of these. Long-term therapy is equally possible as
is adjuvant therapy in the context of other treatment strategies,
as described above.
[0098] In certain embodiments of such methods, one or more
polypeptide therapeutic agents can be administered, together
(simultaneously) or at different times (sequentially). In addition,
polypeptide therapeutic agents can be administered with another
type of compounds for treating cancer or for inhibiting
angiogenesis.
[0099] In certain embodiments, the subject anti-IGF1R antibodies
agents of the invention can be used alone. Alternatively, the
subject agents may be used in combination with other conventional
anti-cancer therapeutic approaches directed to treatment or
prevention of proliferative disorders (e.g., tumor). For example,
such methods can be used in prophylactic cancer prevention,
prevention of cancer recurrence and metastases after surgery, and
as an adjuvant of other conventional cancer therapy. The present
disclosure recognizes that the effectiveness of conventional cancer
therapies (e.g., chemotherapy, radiation therapy, phototherapy,
immunotherapy, and surgery) can be enhanced through the use of a
subject polypeptide therapeutic agent.
[0100] A wide array of conventional compounds have been shown to
have anti-neoplastic activities. These compounds have been used as
pharmaceutical agents in chemotherapy to shrink solid tumors,
prevent metastases and further growth, or decrease the number of
malignant cells in leukemic or bone marrow malignancies. Although
chemotherapy has been effective in treating various types of
malignancies, many anti-neoplastic compounds induce undesirable
side effects. It has been shown that when two or more different
treatments are combined, the treatments may work synergistically
and allow reduction of dosage of each of the treatments, thereby
reducing the detrimental side effects exerted by each compound at
higher dosages. In other instances, malignancies that are
refractory to a treatment may respond to a combination therapy of
two or more different treatments.
[0101] When a polypeptide therapeutic agent of the present
invention is administered in combination with another conventional
anti-neoplastic agent, either concomitantly or sequentially, such
therapeutic agent may be found to enhance the therapeutic effect of
the anti-neoplastic agent or overcome cellular resistance to such
anti-neoplastic agent. This allows decrease of dosage of an
anti-neoplastic agent, thereby reducing the undesirable side
effects, or restores the effectiveness of an anti-neoplastic agent
in resistant cells.
[0102] Pharmaceutical compounds that may be used for combinatory
anti-tumor therapy include, merely to illustrate:
aminoglutethimide, amsacrine, anastrozole, asparaginase, bcg,
bicalutamide, bleomycin, buserelin, busulfan, campothecin,
capecitabine, carboplatin, carmustine, chlorambucil, cisplatin,
cladribine, clodronate, colchicine, cyclophosphamide, cyproterone,
cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol,
diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol,
estramustine, etoposide, exemestane, filgrastim, fludarabine,
fludrocortisone, fluorouracil, fluoxymesterone, flutamide,
gemcitabine, genistein, goserelin, hydroxyurea, idarubicin,
ifosfamide, imatinib, interferon, irinotecan, ironotecan,
letrozole, leucovorin, leuprolide, levamisole, lomustine,
mechlorethamine, medroxyprogesterone, megestrol, melphalan,
mercaptopurine, mesna, methotrexate, mitomycin, mitotane,
mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin,
paclitaxel, pamidronate, pentostatin, plicamycin, porfimer,
procarbazine, raltitrexed, rituximab, streptozocin, suramin,
tamoxifen, temozolomide, teniposide, testosterone, thioguanine,
thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin,
vinblastine, vincristine, vindesine, and vinorelbine.
[0103] Certain chemotherapeutic anti-tumor compounds may be
categorized by their mechanism of action into, for example,
following groups: anti-metabolites/anti-cancer agents, such as
pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine,
gemcitabine and cytarabine) and purine analogs, folate antagonists
and related inhibitors (mercaptopurine, thioguanine, pentostatin
and 2-chlorodeoxyadenosine (cladribine));
antiproliferative/antimitotic agents including natural products
such as vinca alkaloids (vinblastine, vincristine, and
vinorelbine), microtubule disruptors such as taxane (paclitaxel,
docetaxel), vincristin, vinblastin, nocodazole, epothilones and
navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA
damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin,
busulfan, camptothecin, carboplatin, chlorambucil, cisplatin,
cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin,
epirubicin, hexamethylmelamineoxaliplatin, iphosphamide, melphalan,
merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin,
procarbazine, taxol, taxotere, teniposide,
triethylenethiophosphoramide and etoposide (VP16)); antibiotics
such as dactinomycin (actinomycin D), daunorubicin, doxorubicin
(adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins,
plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase
which systemically metabolizes L-asparagine and deprives cells
which do not have the capacity to synthesize their own asparagine);
antiplatelet agents; antiproliferative/antimitotic alkylating
agents such as nitrogen mustards (mechlorethamine, cyclophosphamide
and analogs, melphalan, chlorambucil), ethylenimines and
methylmelamines (hexamethylmelamine and thiotepa), alkyl
sulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs,
streptozocin), trazenes--dacarbazinine (DTIC);
antiproliferative/antimitotic antimetabolites such as folic acid
analogs (methotrexate); platinum coordination complexes (cisplatin,
carboplatin), procarbazine, hydroxyurea, mitotane,
aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen,
goserelin, bicalutamide, nilutamide) and aromatase inhibitors
(letrozole, anastrozole); anticoagulants (heparin, synthetic
heparin salts and other inhibitors of thrombin); fibrinolytic
agents (such as tissue plasminogen activator, streptokinase and
urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel,
abciximab; antimigratory agents; antisecretory agents (breveldin);
immunosuppressives (cyclosporine, tacrolimus (FK-506), sirolimus
(rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic
compounds (TNP-470, genistein) and growth factor inhibitors (e.g.,
VEGF inhibitors, fibroblast growth factor (FGF) inhibitors);
angiotensin receptor blocker; nitric oxide donors; anti-sense
oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors
and differentiation inducers (tretinoin); mTOR inhibitors,
topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine,
camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin,
etoposide, idarubicin and mitoxantrone, topotecan, irinotecan),
corticosteroids (cortisone, dexamethasone, hydrocortisone,
methylpednisolone, prednisone, and prenisolone); growth factor
signal transduction kinase inhibitors; mitochondrial dysfunction
inducers and caspase activators; and chromatin disruptors.
[0104] Depending on the nature of the combinatory therapy,
administration of the polypeptide therapeutic agents may be
continued while the other therapy is being administered and/or
thereafter. Administration of the polypeptide therapeutic agents
may be made in a single dose, or in multiple doses. In some
instances, administration of the polypeptide therapeutic agents is
commenced at least several days prior to the conventional therapy,
while in other instances, administration is begun either
immediately before or at the time of the administration of the
conventional therapy.
[0105] In one example of a diagnostic application, a biological
sample, such as serum or a tissue biopsy, from a patient suspected
of having a condition characterized by inappropriate angiogenesis
is contacted with a detectably labeled polypeptide of the
disclosure to detect levels of IGF1R. The levels of IGF1R detected
are then compared to levels of IGF1R detected in a normal sample
also contacted with the labeled polypeptide. An increase of at
least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% in the levels
of the IGF1R may be considered a diagnostic indicator.
[0106] In certain embodiments, the IGF1R binding polypeptides are
further attached to a label that is able to be detected (e.g., the
label can be a radioisotope, fluorescent compound, enzyme or enzyme
co-factor). The active moiety may be a radioactive agent, such as:
radioactive heavy metals such as iron chelates, radioactive
chelates of gadolinium or manganese, positron emitters of oxygen,
nitrogen, iron, carbon, or gallium, .sup.43K, .sup.52Fe, .sup.57Co,
.sup.67Cu, .sup.67Ga, .sup.68Ga, .sup.123I, .sup.125I, .sup.131I,
.sup.132I, or .sup.99Tc. A binding agent affixed to such a moiety
may be used as an imaging agent and is administered in an amount
effective for diagnostic use in a mammal such as a human and the
localization and accumulation of the imaging agent is then
detected. The localization and accumulation of the imaging agent
may be detected by radioscintigraphy, nuclear magnetic resonance
imaging, computed tomography or positron emission tomography.
Immunoscintigraphy using IGF1R binding polypeptides directed at
IGF1R may be used to detect and/or diagnose cancers and
vasculature. For example, any of the binding polypeptide against a
IGF1R marker labeled with .sup.99Technetium, .sup.111Indium, or
.sup.125Iodine may be effectively used for such imaging. As will be
evident to the skilled artisan, the amount of radioisotope to be
administered is dependent upon the radioisotope. Those having
ordinary skill in the art can readily formulate the amount of the
imaging agent to be administered based upon the specific activity
and energy of a given radionuclide used as the active moiety.
Typically 0.1-100 millicuries per dose of imaging agent, preferably
1-10 millicuries, most often 2-5 millicuries are administered.
Thus, compositions according to the present invention useful as
imaging agents comprising a targeting moiety conjugated to a
radioactive moiety comprise 0.1-100 millicuries, in some
embodiments preferably 1-10 millicuries, in some embodiments
preferably 2-5 millicuries, in some embodiments more preferably 1-5
millicuries.
[0107] The IGF1R binding polypeptides can also be used to deliver
additional therapeutic agents (including but not limited to drug
compounds, chemotherapeutic compounds, and radiotherapeutic
compounds) to a cell or tissue expressing IGF1R. In one example,
the IGF1R binding polypeptide is fused to a chemotherapeutic agent
for targeted delivery of the chemotherapeutic agent to a tumor cell
or tissue expressing IGF1R.
[0108] The IGF1R binding polypeptides are useful in a variety of
applications, including research, diagnostic and therapeutic
applications. For instance, they can be used to isolate and/or
purify receptor or portions thereof, and to study receptor
structure (e.g., conformation) and function.
[0109] In certain aspects, the various binding polypeptides can be
used to detect or measure the expression of IGF1R, for example, on
endothelial cells (e.g., venous endothelial cells), or on cells
transfected with a IGF1R gene. Thus, they also have utility in
applications such as cell sorting and imaging (e.g., flow
cytometry, and fluorescence activated cell sorting), for diagnostic
or research purposes.
[0110] In certain embodiments, the binding polypeptides of
fragments thereof can be labeled or unlabeled for diagnostic
purposes. Typically, diagnostic assays entail detecting the
formation of a complex resulting from the binding of a binding
polypeptide to IGF1R. The binding polypeptides or fragments can be
directly labeled, similar to antibodies. A variety of labels can be
employed, including, but not limited to, radionuclides,
fluorescers, enzymes, enzyme substrates, enzyme cofactors, enzyme
inhibitors and ligands (e.g., biotin, haptens). Numerous
appropriate immunoassays are known to the skilled artisan (U.S.
Pat. Nos. 3,817,827; 3,850,752; 3,901,654; and 4,098,876). When
unlabeled, the binding polypeptides can be used in assays, such as
agglutination assays. Unlabeled binding polypeptides can also be
used in combination with another (one or more) suitable reagent
which can be used to detect the binding polypeptide, such as a
labeled antibody reactive with the binding polypeptide or other
suitable reagent (e.g., labeled protein A).
[0111] In one embodiment, the binding polypeptides of the present
invention can be utilized in enzyme immunoassays, wherein the
subject polypeptides are conjugated to an enzyme. When a biological
sample comprising an IGF1R protein is combined with the subject
binding polypeptides, binding occurs between the binding
polypeptides and the IGF1R protein. In one embodiment, a sample
containing cells expressing an IGF1R protein (e.g., endothelial
cells) is combined with the subject antibodies, and binding occurs
between the binding polypeptides and cells bearing an IGF1R protein
recognized by the binding polypeptide. These bound cells can be
separated from unbound reagents and the presence of the binding
polypeptide-enzyme conjugate specifically bound to the cells can be
determined, for example, by contacting the sample with a substrate
of the enzyme which produces a color or other detectable change
when acted on by the enzyme. In another embodiment, the subject
binding polypeptides can be unlabeled, and a second, labeled
polypeptide (e.g., an antibody) can be added which recognizes the
subject binding polypeptide.
[0112] In certain aspects, kits for use in detecting the presence
of an IGF1R protein in a biological sample can also be prepared.
Such kits will include an IGF1R binding polypeptide which binds to
a IGF1R protein or portion of said receptor, as well as one or more
ancillary reagents suitable for detecting the presence of a complex
between the binding polypeptide and the receptor protein or
portions thereof. The polypeptide compositions of the present
invention can be provided in lyophilized form, either alone or in
combination with additional antibodies specific for other epitopes.
The binding polypeptides and/or antibodies, which can be labeled or
unlabeled, can be included in the kits with adjunct ingredients
(e.g., buffers, such as Tris, phosphate and carbonate, stabilizers,
excipients, biocides and/or inert proteins, e.g., bovine serum
albumin). For example, the binding polypeptides and/or antibodies
can be provided as a lyophilized mixture with the adjunct
ingredients, or the adjunct ingredients can be separately provided
for combination by the user. Generally these adjunct materials will
be present in less than about 5% weight based on the amount of
active binding polypeptide or antibody, and usually will be present
in a total amount of at least about 0.001% weight based on
polypeptide or antibody concentration. Where a second antibody
capable of binding to the binding polypeptide is employed, such
antibody can be provided in the kit, for instance in a separate
vial or container. The second antibody, if present, is typically
labeled, and can be formulated in an analogous manner with the
antibody formulations described above.
[0113] Similarly, the present disclosure also provides a method of
detecting and/or quantitating expression of IGF1R, wherein a
composition comprising a cell or fraction thereof (e.g., membrane
fraction) is contacted with a binding polypeptide which binds to a
IGF1R or portion of the receptor under conditions appropriate for
binding thereto, and the binding is monitored. Detection of the
binding polypeptide, indicative of the formation of a complex
between binding polypeptide and IGF1R or a portion thereof,
indicates the presence of the receptor. Binding of a polypeptide to
the cell can be determined by standard methods, such as those
described in the working examples. The method can be used to detect
expression of IGF1R on cells from an individual. Optionally, a
quantitative expression of IGF1R on the surface of endothelial
cells can be evaluated, for instance, by flow cytometry, and the
staining intensity can be correlated with disease susceptibility,
progression or risk.
[0114] The present disclosure also provides a method of detecting
the susceptibility of a mammal to certain diseases. To illustrate,
the method can be used to detect the susceptibility of a mammal to
diseases which progress based on the amount of IGF1R present on
cells and/or the number of IGF1R-positive cells in a mammal.
[0115] Polypeptide sequences are indicated using standard one- or
three-letter abbreviations. Unless otherwise indicated, each
polypeptide sequence has amino termini at the left and a carboxy
termini at the right; each single-stranded nucleic acid sequence,
and the top strand of each double-stranded nucleic acid sequence,
has a 5' termini at the left and a 3' termini at the right. A
particular polypeptide sequence also can be described by explaining
how it differs from a reference sequence.
[0116] The following terms, unless otherwise indicated, shall be
understood to have the following meanings:
[0117] The terms "IGF1R inhibitor" and "IGF1R antagonist" are used
interchangeably. Each is a molecule that detectably inhibits at
least one function of IGF1R. Conversely, an "IGF1R agonist" is a
molecule that detectably increases at least one function of IGF1R.
The inhibition caused by an IGF1R inhibitor need not be complete so
long as it is detectable using an assay. Any assay of a function of
IGF1R can be used, examples of which are provided herein. Examples
of functions of IGF1R that can be inhibited by an IGF1R inhibitor,
or increased by an IGF1Ragonist, include cancer cell growth or
apoptosis (programmed cell death), and so on. Examples of types of
IGF1R inhibitors and IGF1R agonists include, but are not limited
to, IGF1R binding polypeptides such as antigen binding proteins
(e.g., IGF1R inhibiting antigen binding proteins), antibodies,
antibody fragments, and antibody derivatives.
[0118] The terms "peptide," "polypeptide" and "protein" each refers
to a molecule comprising two or more amino acid residues joined to
each other by peptide bonds. These terms encompass, e.g., native
and artificial proteins, protein fragments and polypeptide analogs
(such as muteins, variants, and fusion proteins) of a protein
sequence as well as post-translationally, or otherwise covalently
or non-covalently, modified proteins. A peptide, polypeptide, or
protein may be monomeric or polymeric.
[0119] A "variant" of a polypeptide (for example, an antibody)
comprises an amino acid sequence wherein one or more amino acid
residues are inserted into, deleted from and/or substituted into
the amino acid sequence relative to another polypeptide sequence.
Disclosed variants include, for example, fusion proteins.
[0120] A "derivative" of a polypeptide is a polypeptide (e.g., an
antibody) that has been chemically modified, e.g., via conjugation
to another chemical moiety (such as, for example, polyethylene
glycol or albumin, e.g., human serum albumin), phosphorylation, and
glycosylation. Unless otherwise indicated, the term "antibody"
includes, in addition to antibodies comprising two full-length
heavy chains and two full-length light chains, derivatives,
variants, fragments, and muteins thereof, examples of which are
described below.
[0121] An "antigen binding protein" is a protein comprising a
portion that binds to an antigen and, optionally, a scaffold or
framework portion that allows the antigen binding portion to adopt
a conformation that promotes binding of the antigen binding protein
to the antigen. Examples of antigen binding proteins include
antibodies, antibody fragments (e.g., an antigen binding portion of
an antibody), antibody derivatives, and antibody analogs. The
antigen binding protein can comprise, for example, an alternative
protein scaffold or artificial scaffold with grafted CDRs or CDR
derivatives. Such scaffolds include, but are not limited to,
antibody-derived scaffolds comprising mutations introduced to, for
example, stabilize the three-dimensional structure of the antigen
binding protein as well as wholly synthetic scaffolds comprising,
for example, a biocompatible polymer. In addition, peptide antibody
mimetics ("PAMs") can be used, as well as scaffolds based on
antibody mimetics utilizing fibronection components as a
scaffold.
[0122] An antigen binding protein can have, for example, the
structure of a naturally occurring immunoglobulin. An
"immunoglobulin" is a tetrameric molecule. In a naturally occurring
immunoglobulin, each tetramer is composed of two identical pairs of
polypeptide chains, each pair having one "light" (about 25 kDa) and
one "heavy" chain (about 50-70 kDa). The amino-terminal portion of
each chain includes a variable region of about 100 to 110 or more
amino acids primarily responsible for antigen recognition. The
carboxy-terminal portion of each chain defines a constant region
primarily responsible for effector function. Human light chains are
classified as kappa or lambda light chains. Heavy chains are
classified as mu, delta, gamma, alpha, or epsilon, and define the
antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
Preferably, the anti-IGF1R antibodies disclosed herein are
characterized by their variable domain region sequences in the
heavy V.sub.H and light V.sub.L amino acid sequences. The preferred
antibody is A6 which is a kappa IgG antibody. Within light and
heavy chains, the variable and constant regions are joined by a "J"
region of about 12 or more amino acids, with the heavy chain also
including a "D" region of about 10 more amino acids. See generally,
Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press,
N.Y. (1989)). The variable regions of each light/heavy chain pair
form the antibody binding site such that an intact immunoglobulin
has two binding sites.
[0123] A "multi-specific antibody" is an antibody that recognizes
more than one epitope on one or more antigens. A subclass of this
type of antibody is a "bi-specific antibody" which recognizes two
distinct epitopes on the same or different antigens.
[0124] An antigen binding protein "specifically binds" to an
antigen (e.g., human IGF1R) if it binds to the antigen with a
dissociation constant of 1 nanomolar or less.
[0125] An "antigen binding domain, "antigen binding region," or
"antigen binding site" is a portion of an antigen binding protein
that contains amino acid residues (or other moieties) that interact
with an antigen and contribute to the antigen binding protein's
specificity and affinity for the antigen. For an antibody that
specifically binds to its antigen, this will include at least part
of at least one of its CDR domains.
[0126] An "epitope" is the portion of a molecule that is bound by
an antigen binding protein (e.g., by an antibody). An epitope can
comprise non-contiguous portions of the molecule (e.g., in a
polypeptide, amino acid residues that are not contiguous in the
polypeptide's primary sequence but that, in the context of the
polypeptide's tertiary and quaternary structure, are near enough to
each other to be bound by an antigen binding protein).
[0127] The "percent homology" of two polynucleotide or two
polypeptide sequences is determined by comparing the sequences
using the GAP computer program (a part of the GCG Wisconsin
Package, version 10.3 (Accelrys, San Diego, Calif.)) using its
default parameters.
[0128] A "host cell" is a cell that can be used to express a
nucleic acid. A host cell can be a prokaryote, for example, E.
coli, or it can be a eukaryote, for example, a single-celled
eukaryote (e.g., a yeast or other fungus), a plant cell (e.g., a
tobacco or tomato plant cell), an animal cell (e.g., a human cell,
a monkey cell, a hamster cell, a rat cell, a mouse cell, or an
insect cell) or a hybridoma. Examples of host cells include the
COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al.,
1981, Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL 163),
Chinese hamster ovary (CHO) cells or their derivatives such as
Veggie CHO and related cell lines which grow in serum-free media
(Rasmussen et al., 1998, Cytotechnology 28:31) or CHO strain
DX-B11, which is deficient in DHFR (Urlaub et al., 1980, Proc.
Natl. Acad. Sci. USA 77:4216-20), HeLa cells, BHK (ATCC CRL 10)
cell lines, the CV1/EBNA cell line derived from the African green
monkey kidney cell line CV1 (ATCC CCL 70) (McMahan et al., 1991,
EMBO J. 10:2821), human embryonic kidney cells such as 293,293 EBNA
or MSR 293, human epidermal A431 cells, human Colo205 cells, other
transformed primate cell lines, normal diploid cells, cell strains
derived from in vitro culture of primary tissue, primary explants,
HL-60, U937, HaK or Jurkat cells. Typically, a host cell is a
cultured cell that can be transformed or transfected with a
polypeptide-encoding nucleic acid, which can then be expressed in
the host cell. The phrase "recombinant host cell" can be used to
denote a host cell that has been transformed or transfected with a
nucleic acid to be expressed. A host cell also can be a cell that
comprises the nucleic acid but does not express it at a desired
level unless a regulatory sequence is introduced into the host cell
such that it becomes operably linked with the nucleic acid. It is
understood that the term host cell refers not only to the
particular subject cell but also to the progeny or potential
progeny of such a cell. Because certain modifications may occur in
succeeding generations due to, e.g., mutation or environmental
influence, such progeny may not, in fact, be identical to the
parent cell, but are still included within the scope of the term as
used herein.
Antigen Binding Proteins
[0129] Antigen binding proteins (e.g., antibodies, antibody
fragments, antibody derivatives, antibody muteins, and antibody
variants) are polypeptides that bind to IGF1R, (preferably, human
IGF1R). Antigen binding proteins include antigen binding proteins
that inhibit a biological activity of IGF1R.
[0130] Oligomers that contain one or more antigen binding proteins
may be employed as IGF1R antagonists. Oligomers may be in the form
of covalently-linked or non-covalently-linked dimers, trimers, or
higher oligomers. Oligomers comprising two or more antigen binding
protein are contemplated for use, with one example being a
homodimer. Other oligomers include heterodimers, homotrimers,
heterotrimers, homotetramers, heterotetramers, etc.
[0131] One embodiment is directed to oligomers comprising multiple
antigen binding proteins joined via covalent or non-covalent
interactions between peptide moieties fused to the antigen binding
proteins. Such peptides may be peptide linkers (spacers), or
peptides that have the property of promoting oligomerization.
Leucine zippers and certain polypeptides derived from antibodies
are among the peptides that can promote oligomerization of antigen
binding proteins attached thereto, as described in more detail
below.
[0132] In particular embodiments, the oligomers comprise from two
to four antigen binding proteins. The antigen binding proteins of
the oligomer may be in any form, such as any of the forms described
above, e.g., variants or fragments. Preferably, the oligomers
comprise antigen binding proteins that have IGF1R binding
activity.
[0133] In one embodiment, an oligomer is prepared using
polypeptides derived from immunoglobulins. Preparation of Fusion
Proteins Comprising Certain Heterologous Polypeptides Fused to
Various Portions of antibody-derived polypeptides (including the Fc
domain) has been described, e.g., by Ashkenazi et al., 1991, Proc.
Natl. Acad. Sci. USA 88:10535; Byrn et al., 1990, Nature 344:677;
and Hollenbaugh et al., 1992 "Construction of Immunoglobulin Fusion
Proteins", in Current Protocols in Immunology, Suppl. 4, pages
10.19.1-10.19.11.
[0134] One embodiment is directed to a dimer comprising two fusion
proteins created by fusing an IGF1R binding fragment of an
anti-IGF1R antibody to the Fc region of an antibody. The dimer can
be made by, for example, inserting a gene fusion encoding the
fusion protein into an appropriate expression vector, expressing
the gene fusion in host cells transformed with the recombinant
expression vector, and allowing the expressed fusion protein to
assemble much like antibody molecules, whereupon interchain
disulfide bonds form between the Fc moieties to yield the
dimer.
[0135] The term "Fc polypeptide" includes native and mutein forms
of polypeptides derived from the Fc region of an antibody.
Truncated forms of such polypeptides containing the hinge region
that promotes dimerization also are included. Fusion proteins
comprising Fc moieties (and oligomers formed therefrom) offer the
advantage of facile purification by affinity chromatography over
Protein A or Protein G columns.
[0136] Another method for preparing oligomeric antigen binding
proteins involves use of a leucine zipper. Leucine zipper domains
are peptides that promote oligomerization of the proteins in which
they are found. Leucine zippers were originally identified in
several DNA-binding proteins (Landschulz et al., 1988, Science
240:1759), and have since been found in a variety of different
proteins. Among the known leucine zippers are naturally occurring
peptides and derivatives thereof that dimerize or trimerize.
Examples of leucine zipper domains suitable for producing soluble
oligomeric proteins are described in WO 94/10308, and the leucine
zipper derived from lung surfactant protein D (SPD) described in
Hoppe et al., 1994, FEBS Letters 344:191. The use of a modified
leucine zipper that allows for stable trimerization of a
heterologous protein fused thereto is described in Fanslow et al.,
1994, Semin. Immunol. 6:267-78. In one approach, recombinant fusion
proteins comprising an anti-IGF1R antibody fragment or derivative
fused to a leucine zipper peptide are expressed in suitable host
cells, and the soluble oligomeric anti-IGF1R antibody fragments or
derivatives that form are recovered from the culture
supernatant.
[0137] Antigen-binding fragments of antigen binding proteins of the
invention may be produced by conventional techniques. Examples of
such fragments include, but are not limited to, Fab and
F(ab').sub.2 fragments.
[0138] The present disclosure provides monoclonal antibodies that
bind to IGF1R. Monoclonal antibodies may be produced using any
technique known in the art, e.g., by immortalizing spleen cells
harvested from the transgenic animal after completion of the
immunization schedule. The spleen cells can be immortalized using
any technique known in the art, e.g., by fusing them with myeloma
cells to produce hybridomas. Myeloma cells for use in
hybridoma-producing fusion procedures preferably are
non-antibody-producing, have high fusion efficiency, and enzyme
deficiencies that render them incapable of growing in certain
selective media which support the growth of only the desired fused
cells (hybridomas). Examples of suitable cell lines for use in
mouse fusions include Sp-20, P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 4
1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0
Bul; examples of cell lines used in rat fusions include R210.RCY3,
Y3-Ag 1.2.3, IR983F and 48210. Other cell lines useful for cell
fusions are U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6.
[0139] Antigen binding proteins directed against IGF1R can be used,
for example, in assays to detect the presence of IGF1R
polypeptides, either in vitro or in vivo. The antigen binding
proteins also may be employed in purifying IGF1R proteins by
immunoaffinity chromatography. Blocking antigen binding proteins
can be used in the methods disclosed herein. Such antigen binding
proteins that function as IGF1R antagonists may be employed in
treating any IGF1R-induced condition, including but not limited to
various cancers.
[0140] Antigen binding proteins may be employed in an in vitro
procedure, or administered in vivo to inhibit IGF1R-induced
biological activity. Disorders caused or exacerbated (directly or
indirectly) by the proteolytic activation of IGF1R, examples of
which are provided herein, thus may be treated. In one embodiment,
the present invention provides a therapeutic method comprising in
vivo administration of a IGF1R blocking antigen binding protein to
a mammal in need thereof in an amount effective for reducing an
IGF1R-induced biological activity.
[0141] Antigen binding proteins include fully human monoclonal
antibodies that inhibit a biological activity of IGF1R.
[0142] Antigen binding proteins may be prepared by any of a number
of conventional techniques. For example, they may be purified from
cells that naturally express them (e.g., an antibody can be
purified from a hybridoma that produces it), or produced in
recombinant expression systems, using any technique known in the
art.
[0143] Any expression system known in the art can be used to make
the recombinant polypeptides of the invention. In general, host
cells are transformed with a recombinant expression vector that
comprises DNA encoding a desired polypeptide. Among the host cells
that may be employed are prokaryotes, yeast or higher eukaryotic
cells. Prokaryotes include gram negative or gram positive
organisms, for example E. coli or bacilli. Higher eukaryotic cells
include insect cells and established cell lines of mammalian
origin. Examples of suitable mammalian host cell lines include the
COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al.,
1981, Cell 23:175), L cells, 293 cells, C127 cells, 3T3 cells (ATCC
CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, BHK (ATCC
CRL 10) cell lines, and the CV1/EBNA cell line derived from the
African green monkey kidney cell line CV1 (ATCC CCL 70) as
described by McMahan et al., 1991, EMBO J. 10: 2821. Appropriate
cloning and expression vectors for use with bacterial, fungal,
yeast, and mammalian cellular hosts are described by Pouwels et al.
(Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., 1985).
[0144] The transformed cells can be cultured under conditions that
promote expression of the polypeptide, and the polypeptide
recovered by conventional protein purification procedures. One such
purification procedure includes the use of affinity chromatography,
e.g., over a matrix having all or a portion (e.g., the
extracellular domain) of IGF1R bound thereto. Polypeptides
contemplated for use herein include substantially homogeneous
recombinant mammalian anti-IGF1R antibody polypeptides
substantially free of contaminating endogenous materials.
[0145] Antigen binding proteins may be prepared, and screened for
desired properties, by any of a number of known techniques. Certain
of the techniques involve isolating a nucleic acid encoding a
polypeptide chain (or portion thereof) of an antigen binding
protein of interest (e.g., an anti-IGF1R antibody), and
manipulating the nucleic acid through recombinant DNA technology.
The nucleic acid may be fused to another nucleic acid of interest,
or altered (e.g., by mutagenesis or other conventional techniques)
to add, delete, or substitute one or more amino acid residues, for
example.
[0146] Single chain antibodies may be formed by linking heavy and
light chain variable domain (Fv region) fragments via an amino acid
bridge (short peptide linker), resulting in a single polypeptide
chain. Such single-chain Fvs (scFvs) have been prepared by fusing
DNA encoding a peptide linker between DNAs encoding the two
variable domain polypeptides (V.sub.L and V.sub.H). The resulting
polypeptides can fold back on themselves to form antigen-binding
monomers, or they can form multimers (e.g., dimers, trimers, or
tetramers), depending on the length of a flexible linker between
the two variable domains (Kortt et al., 1997, Prot. Eng. 10:423;
Kortt et al., 2001, Biomol. Eng. 18:95-108). By combining different
V.sub.L and V.sub.H-comprising polypeptides, one can form
multimeric scFvs that bind to different epitopes (Kriangkum et al.,
2001, Biomol. Eng. 18:31-40). Techniques developed for the
production of single chain antibodies include those described in
U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423; Huston et
al., 1988, Proc. Natl. Acad. Sci. USA 85:5879; Ward et al., 1989,
Nature 334:544, de Graaf et al., 2002, Methods Mol. Biol.
178:379-87.
[0147] Techniques are known for deriving an antibody of a different
subclass or isotype from an antibody of interest, i.e., subclass
switching. Thus, IgG antibodies may be derived from an IgM
antibody, for example, and vice versa. Such techniques allow the
preparation of new antibodies that possess the antigen-binding
properties of a given antibody (the parent antibody), but also
exhibit biological properties associated with an antibody isotype
or subclass different from that of the parent antibody. Recombinant
DNA techniques may be employed. Cloned DNA encoding particular
antibody polypeptides may be employed in such procedures, e.g., DNA
encoding the constant domain of an antibody of the desired isotype
(Lantto et al., 2002, Methods Mol. Biol. 178:303-16). Moreover, if
an IgG4 is desired, it may also be desired to introduce a point
mutation (CPSCP->CPPCP) in the hinge region (Bloom et al., 1997,
Protein Science 6:407) to alleviate a tendency to form intra-H
chain disulfide bonds that can lead to heterogeneity in the IgG4
antibodies.
[0148] In particular embodiments, antigen binding proteins of the
present invention have a binding affinity (K.sub.a) for IGF1R of at
least 10.sup.6. In other embodiments, the antigen binding proteins
exhibit a K.sub.a of at least 10.sup.7, at least 10.sup.8, at least
10.sup.9, or at least 10.sup.10. In another embodiment, the antigen
binding protein exhibits a K.sub.a substantially the same as that
of an antibody described herein in the Examples.
[0149] In another embodiment, the present disclosure provides an
antigen binding protein that has a low dissociation rate from
IGF1R. In one embodiment, the antigen binding protein has a
K.sub.off of 1.times.10.sup.-4 to .sup.-1 or lower. In another
embodiment, the K.sub.off is 5.times.10.sup.-5 to .sup.-1 or lower.
In another embodiment, the K.sub.off is substantially the same as
an antibody described herein. In another embodiment, the antigen
binding protein binds to IGF1R with substantially the same
K.sub.off as an antibody described herein.
[0150] In another aspect, the present disclosure provides an
antigen binding protein that inhibits an activity of IGF1R. In one
embodiment, the antigen binding protein has an IC.sub.50 of 1000 nM
or lower. In another embodiment, the IC.sub.50 is 100 nM or lower;
in another embodiment, the IC.sub.50 is 10 nM or lower. In another
embodiment, the IC.sub.50 is substantially the same as that of an
antibody described herein in the Examples. In another embodiment,
the antigen binding protein inhibits an activity of IGF1R with
substantially the same IC.sub.50 as an antibody described
herein.
[0151] In another aspect, the present disclosure provides an
antigen binding protein that binds to human IGF1R expressed on the
surface of a cell and, when so bound, inhibits IGF1R signaling
activity in the cell without causing a significant reduction in the
amount of IGF1R on the surface of the cell. Any method for
determining or estimating the amount of IGF1R on the surface and/or
in the interior of the cell can be used. In other embodiments,
binding of the antigen binding protein to the IGF1R-expressing cell
causes less than about 75%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, 1%,
or 0.1% of the cell-surface IGF1R to be internalized.
[0152] In another aspect, the present disclosure provides an
antigen binding protein having a half-life of at least one day in
vitro or in vivo (e.g., when administered to a human subject). In
one embodiment, the antigen binding protein has a half-life of at
least three days. In another embodiment, the antigen binding
protein has a half-life of four days or longer. In another
embodiment, the antigen binding protein has a half-life of eight
days or longer. In another embodiment, the antigen binding protein
is derivatized or modified such that it has a longer half-life as
compared to the underivatized or unmodified antigen binding
protein. In another embodiment, the antigen binding protein
contains one or more point mutations to increase serum half-life,
such as described in WO00/09560, incorporated by reference
herein.
[0153] The present disclosure further provides multi-specific
antigen binding proteins, for example, bispecific antigen binding
protein, e.g., antigen binding protein that bind to two different
epitopes of IGF1R, or to an epitope of IGF1R and an epitope of
another molecule, via two different antigen binding sites or
regions. Moreover, bispecific antigen binding protein as disclosed
herein can comprise a IGF1R binding site from one of the
herein-described antibodies and a second IGF1R binding region from
another of the herein-described antibodies, including those
described herein by reference to other publications. Alternatively,
a bispecific antigen binding protein may comprise an antigen
binding site from one of the herein described antibodies and a
second antigen binding site from another IGF1R antibody that is
known in the art, or from an antibody that is prepared by known
methods or the methods described herein.
[0154] Numerous methods of preparing bispecific antibodies are
known in the art. Such methods include the use of hybrid-hybridomas
as described by Milstein et al., 1983, Nature 305:537, and chemical
coupling of antibody fragments (Brennan et al., 1985, Science
229:81; Glennie et al., 1987, J. Immunol. 139:2367; U.S. Pat. No.
6,010,902). Moreover, bispecific antibodies can be produced via
recombinant means, for example by using leucine zipper moieties
(i.e., from the Fos and Jun proteins, which preferentially form
heterodimers; Kostelny et al., 1992, J. Immunol. 148:1547) or other
lock and key interactive domain structures as described in U.S.
Pat. No. 5,582,996. Additional useful techniques include those
described in U.S. Pat. Nos. 5,959,083; and 5,807,706.
[0155] In another aspect, the antigen binding protein comprises a
derivative of an antibody. The derivatized antibody can comprise
any molecule or substance that imparts a desired property to the
antibody, such as increased half-life in a particular use. The
derivatized antibody can comprise, for example, a detectable (or
labeling) moiety (e.g., a radioactive, colorimetric, antigenic or
enzymatic molecule, a detectable bead (such as a magnetic or
electrodense (e.g., gold) bead), or a molecule that binds to
another molecule (e.g., biotin or streptavidin), a therapeutic or
diagnostic moiety (e.g., a radioactive, cytotoxic, or
pharmaceutically active moiety), or a molecule that increases the
suitability of the antibody for a particular use (e.g.,
administration to a subject, such as a human subject, or other in
vivo or in vitro uses). Examples of molecules that can be used to
derivatize an antibody include albumin (e.g., human serum albumin)
and polyethylene glycol (PEG). Albumin-linked and PEGylated
derivatives of antibodies can be prepared using techniques well
known in the art. In one embodiment, the antibody is conjugated or
otherwise linked to transthyretin (TTR) or a TTR variant. The TTR
or TTR variant can be chemically modified with, for example, a
chemical selected from the group consisting of dextran,
poly(n-vinyl pyurrolidone), polyethylene glycols, propropylene
glycol homopolymers, polypropylene oxide/ethylene oxide
co-polymers, polyoxyethylated polyols and polyvinyl alcohols.
[0156] The following terms, unless otherwise indicated, shall be
understood to have the following meanings:
Indications
[0157] In one aspect, the present disclosure provides methods of
treating a subject. The method can, for example, have a generally
salubrious effect on the subject, e.g., it can increase the
subject's expected longevity. Alternatively, the method can, for
example, treat, prevent, cure, relieve, or ameliorate ("treat") a
disease, disorder, condition, or illness ("a condition"). Among the
conditions to be treated are conditions characterized by
inappropriate expression or activity of IGF1R. In some such
conditions, the expression or activity level is too high, and the
treatment comprises administering an IGF1R antagonist as described
herein. The disorders or conditions are cancer-related. In
particular, those cancers include, but are not limited to, lung,
ovarian and colon carcinoma and various myelomas.
[0158] Specific medical conditions and diseases that are treatable
or preventable with the antigen binding proteins of this disclosure
include various cancers.
Therapeutic Methods and Administration of Antigen Binding
Proteins
[0159] Certain methods provided herein comprise administering an
IGF1R binding antigen binding protein to a subject, thereby
reducing an IGF1R-induced biological response that plays a role in
a particular condition. In particular embodiments, methods of the
invention involve contacting endogenous IGF1R with an IGF1R binding
antigen binding protein, e.g., via administration to a subject or
in an ex vivo procedure.
[0160] The term "treatment" encompasses alleviation or prevention
of at least one symptom or other aspect of a disorder, or reduction
of disease severity, and the like. An antigen binding protein need
not effect a complete cure, or eradicate every symptom or
manifestation of a disease, to constitute a viable therapeutic
agent. As is recognized in the pertinent field, drugs employed as
therapeutic agents may reduce the severity of a given disease
state, but need not abolish every manifestation of the disease to
be regarded as useful therapeutic agents. Similarly, a
prophylactically administered treatment need not be completely
effective in preventing the onset of a condition in order to
constitute a viable prophylactic agent. Simply reducing the impact
of a disease (for example, by reducing the number or severity of
its symptoms, or by increasing the effectiveness of another
treatment, or by producing another beneficial effect), or reducing
the likelihood that the disease will occur or worsen in a subject,
is sufficient. One embodiment of the invention is directed to a
method comprising administering to a patient an IGF1R antagonist in
an amount and for a time sufficient to induce a sustained
improvement over baseline of an indicator that reflects the
severity of the particular disorder.
[0161] As is understood in the pertinent field, pharmaceutical
compositions comprising the antibodies and fragments thereof of the
disclosure are administered to a subject in a manner appropriate to
the indication. Pharmaceutical compositions may be administered by
any suitable technique, including but not limited to, parenterally,
topically, or by inhalation. If injected, the pharmaceutical
composition can be administered, for example, via intra-articular,
intravenous, intramuscular, intralesional, intraperitoneal or
subcutaneous routes, by bolus injection, or continuous infusion.
Localized administration, e.g. at a site of disease or injury is
contemplated, as are transdermal delivery and sustained release
from implants. Delivery by inhalation includes, for example, nasal
or oral inhalation, use of a nebulizer, inhalation of the
antagonist in aerosol form, and the like. Other alternatives
include eyedrops; oral preparations including pills, syrups,
lozenges or chewing gum; and topical preparations such as lotions,
gels, sprays, and ointments.
[0162] Use of antigen binding proteins in ex vivo procedures also
is contemplated. For example, a patient's blood or other bodily
fluid may be contacted with an antigen binding protein that binds
IGF1R ex vivo. The antigen binding protein may be bound to a
suitable insoluble matrix or solid support material.
[0163] Advantageously, antigen binding proteins are administered in
the form of a composition comprising one or more additional
components such as a physiologically acceptable carrier, excipient
or diluent. Optionally, the composition additionally comprises one
or more physiologically active agents, for example, a second
inflammation- or immune-inhibiting substance, an anti-angiogenic
substance, an analgesic substance, etc., non-exclusive examples of
which are provided herein. In various particular embodiments, the
composition comprises one, two, three, four, five, or six
physiologically active agents in addition to an IGF1R binding
antigen binding protein
Combination Therapy
[0164] In another aspect, the present disclosure provides a method
of treating a subject with a IGF1R inhibiting antigen binding
protein and one or more other treatments. In one embodiment, such a
combination therapy achieves synergy or an additive effect by, for
example, attacking multiple sites or molecular targets in a tumor.
Types of combination therapies that can be used in connection with
the present invention include inhibiting or activating (as
appropriate) multiple nodes in a single disease-related pathway,
multiple pathways in a target cell, and multiple cell types within
a target tissue.
[0165] In another embodiment, a combination therapy method
comprises administering to the subject two, three, four, five, six,
or more of the IGF1R agonists or antagonists described herein. In
another embodiment, the method comprises administering to the
subject two or more treatments that together inhibit or activate
(directly or indirectly) IGF1R-mediated signal transduction.
Examples of such methods include using combinations of two or more
IGF1R inhibiting antigen binding proteins, of a IGF1R inhibiting
antigen binding protein and one or more other therapeutic moiety
having anti-cancer properties (for example, cytotoxic agents,
and/or immunomodulators), or of a IGF1R inhibiting antigen binding
protein and one or more other treatments (e.g., surgery, or
radiation). Furthermore, one or more anti-IGF1R antibodies or
antibody derivatives can be used in combination with one or more
molecules or other treatments, wherein the other molecule(s) and/or
treatment(s) do not directly bind to or affect IGF1R, but which
combination is effective for treating or preventing the condition
being treated. In one embodiment, one or more of the molecule(s)
and/or treatment(s) treats or prevents a condition that is caused
by one or more of the other molecule(s) or treatment(s) in the
course of therapy, e.g., nausea, fatigue, alopecia, cachexia,
insomnia, etc. In every case where a combination of molecules
and/or other treatments is used, the individual molecule(s) and/or
treatment(s) can be administered in any order, over any length of
time, which is effective, e.g., simultaneously, consecutively, or
alternately. In one embodiment, the method of treatment comprises
completing a first course of treatment with one molecule or other
treatment before beginning a second course of treatment. The length
of time between the end of the first course of treatment and
beginning of the second course of treatment can be any length of
time that allows the total course of therapy to be effective, e.g.,
seconds, minutes, hours, days, weeks, months, or even years.
[0166] In another embodiment, the method comprises administering
one or more of the IGF1R antagonists described herein and one or
more other treatments (e.g., a therapeutic or palliative
treatment). Where a method comprises administering more than one
treatment to a subject, it is to be understood that the order,
timing, number, concentration, and volume of the administrations is
limited only by the medical requirements and limitations of the
treatment, i.e., two treatments can be administered to the subject,
e.g., simultaneously, consecutively, alternately, or according to
any other regimen.
Example 1
[0167] This example illustrates in vitro data for anti-IGF1R
antibodies cellular binding EC.sub.50 measurements. This example
shows the binding characteristic for these antibodies in terms of
the maximal cell binding and the concentration at which 50% binding
saturation (EC.sub.50) is reached. In this example, the
experimental procedure is as follows: 50,000 MCF7 breast cancer
cells were aliquoted into the wells of a 96-well, v-bottom plate in
100 .mu.l FACS Buffer (PBS+2% FBS). A dilution curve of antibodies
was made in FACS Buffer encompassing the concentrations shown in
FIG. 3. Cells were spun down, washed 1.times. with FACS Buffer, and
then resuspended in 25 .mu.l of antibody solution in triplicate.
After 0.5 hr incubation, cells were washed 1.times. with FACS
Buffer and resuspended in 50 .mu.l PE-conjugated, goat anti-human
IgG (.gamma.-chain specific) secondary antibody (Southern Biotech
Cat #2040-09). Cells were further incubated for 0.5 hr and then
washed 1.times. with FACS Buffer. Cells were resuspended in 25
.mu.l FACS Buffer and the median fluorescence intensity in the
FL2-H channel was determined using the Intellicyt HTFC flow
cytometer.
[0168] Results: As shown in FIG. 3, the cell binding EC.sub.50 for
these anti-IGF1R antibodies on MCF7 cells was ranged from 3.7 .mu.M
(B9) to 19.5 nM (C2). While these values range over orders of
magnitude, all antibodies show strong, specific binding to MCF7
cells expressing IGF1R. Data was collected on the Intellicyt HTFC
flow cytometer, processed using FlowJo software, and analyzed and
plotted in Graph Pad Prizm using non-linear regression fit. Data
points are shown as the median fluorescence intensity (MFI) of
positively labeled cells +/- Std Error.
Example 2
[0169] This example illustrates in vitro data showing IGF1
stimulated auto-phosphorylation of IGF1R in MCF7 breast cancer
cells. This example demonstrates the ability of antibodies to block
the activation of and therefore the function of IGF1R in cancer
cells. Protocol: 40,000 MCF7 cells were plated in the wells of a
96-well cell culture cluster in 100 .mu.l Phenol Red-free DMEM
media supplemented with 10% FBS. 24 hr later, media were removed
and the cells washed 1.times. with PBS, and then starved for 18 hr
in 100 ul starvation media (Phenol Red-free DMEM+0% FBS).
Antibodies were diluted to 20 .mu.g/ml (2.times. final
concentration) in 50 .mu.l serum-free media, then added to the
cells after removal of starvation media. After 3 hr incubation, 50
.mu.l of 100 ng/ml IGF1 was added to the cells for a final
concentration of 50 ng/ml. Cells were then incubated for 5 min.
Cells were washed with PBS and lysed in 1.times. Cell Lysis Buffer
(Cell Signaling). Phosphorylation of IGF1R was detected using
PathScan Phospho-IGF-1 Receptor .beta. (Tyr1131) Sandwich ELISA
according to the manufacturer's protocol (Cell Signaling) adjusted
for half area ELISA plates.
[0170] Results: As shown in FIG. 5, MCF7 cells treated with 50
ng/ml IGF1 showed robust activating auto-phosphorylation of IGF1R
(column 2, +, compared to column 1, -). Pre-treatment of cells with
anti-IGF1R antibodies variably blocks this activation of IGF1R.
Clones A6, C2, B9, B10, B10VAR, and C8 showed the most potent
antagonism of IGF1R auto-phosphorylation indicating these clones
are potential candidates for therapeutic intervention against IGF1R
in cancer. Data shown as the absorption at 450 nm (ABS 450 nm) of
triplicate samples +/- Std Error and is directly proportional to
IGF1R phosphorylation/activation.
Example 3
[0171] This example illustrates in vitro data showing IGF1
stimulated auto-phosphorylation of IGF1R in MCF7 breast cancer
cells. Specifically, this example demonstrates the IC.sub.50
(concentration at half maximum inhibition) for the anti-IGF1R
antibodies blocking this auto-phosphorylation. This example
suggests the efficacy of anti-IGF1R antibodies in blocking IGF1R
function in vitro. Protocol: 40,000 MCF7 cells were plated in the
wells of a 96-well cell culture cluster in 100 .mu.l Phenol
Red-free DMEM media supplemented with 10% FBS. 24 hr later, media
were removed and the cells washed 1.times. with PBS, and then
starved for 24 hr in 100 .mu.l starvation media (Phenol Red-free
DMEM+0% FBS). Antibodies were serially diluted to 2.times. the
desired final concentration in 50 .mu.l serum-free media, then
added to the cells after removal of starvation media. After 10 min
incubation, 50 .mu.l of 100 ng/ml IGF1 was added to the wells for a
final concentration of 50 ng/ml. Cells were then incubated for 5
min. Cells were washed with PBS+0.1% sodium vanadate and lysed in
1.times. Cell Lysis Buffer (Cell Signaling). Phosphorylation of
IGF1R was detected using PathScan Phospho-IGF-1 Receptor .beta.
(Tyr1131) Sandwich ELISA according to the manufacturer's protocol
(Cell Signaling) adjusted for half area ELISA plates.
[0172] Results: As shown in FIG. 6, pre-treatment of cells with
anti-IGF1R antibodies variably blocks the activation of IGF1R. The
B9 clone showed the most potent antagonism of IGF1R with an
IC.sub.50 value of 94 pM. Data shown as the absorption at 450 nm
(ABS 450 nm) of triplicate samples +/- Std Error. Data was analyzed
and plotted in Graph Pad Prizm using non-linear regression fit to
determine IC.sub.50 values.
Example 4
[0173] This example illustrates in vitro data showing the
inhibition of IGF1-stimulated cell proliferation by anti-IGF1R
antibodies. Uncontrolled cell proliferation is a hallmark of cancer
and the ability to inhibit proliferation in IGF1R positive cancer
cells with anti-IGF1R antibodies is requisite for a therapeutic
compound. In this example, 5000 MCF7 breast cancer cells were
plated into the wells of a 96-well cell culture cluster in 100
.mu.l Phenol Red-free DMEM supplemented with 10% FBS, in
triplicate. 24 hr later, media was removed, cells washed 1.times.
with PBS, and 50 serum free media with 20, 2, or 0.2 .mu.g/ml
(2.times. final concentration) anti-IGF1R antibody was added to the
cells. After 0.5 hr incubation, IGF1 was added at a concentration
of 100 ng/ml in 50 (final concentration of IGF1 is 50 ng/ml). Cells
were then incubated for 72 hr, after which the Promega Cell Titer
96 Non-radioactive Cell Proliferation Assay kit was used to
evaluate proliferation. The proliferative index was calculated as
the OD570 of IGF1 treated sample (with or without antibody
treatment)/cells alone.
[0174] Results: As shown in FIG. 7, the anti-IGF1R antibodies C2, B
10, and C8 inhibited IGF1-stimulated MCF7 proliferation in a
dose-dependent manner. Data shown is the mean proliferative index
calculated as the OD 570 of IGF1 treated sample (with or without
antibody treatment)/OD 570 of cells alone of triplicate samples +/-
Std Error.
Example 8
[0175] This example illustrates in vitro data showing
IGF2-stimulated phosphorylation of IGF1R in MCF7 breast cancer
cells. This example demonstrates the ability of antibodies to block
the activation of and therefore the function of IGF1R in cancer
cells. In this example MCF7 cells were plated in the wells of a
96-well cell culture cluster in Phenol Red-free DMEM media
supplemented with 10% FBS. 24 hr later, media were removed and the
cells washed 1.times. with PBS, and then starved for 18 hr in
starvation media (Phenol Red-free DMEM+0% FBS). Antibodies were
diluted to 20 .mu.g/ml (2.times. final concentration) in serum-free
media, then added to the cells after removal of starvation media.
After 0.5 hr incubation, 200 ng/ml IGF2 (2.times. final
concentration) was added to the cells for a final concentration of
100 ng/ml. Cells were then incubated for 5 min. Cells were washed
with PBS and lysed in 1.times. Cell Lysis Buffer (Cell Signaling).
Phosphorylation of IGF1R was detected using PathScan Phospho-IGF-1
Receptor .beta. (Tyr1131) Sandwich ELISA according to the
manufacturer's protocol (Cell Signaling) adjusted for half area
ELISA plates.
[0176] Results: As shown in FIG. 8, MCF7 cells treated with 100
ng/ml IGF2 showed robust activating phosphorylation of IGF1R
(column 2, IGF2 Alone, compared to column 1, Untreated).
Pre-treatment of cells with anti-IGF1R antibodies variably blocked
this activation of IGF1R. Clone B 10 showed the most potent
antagonism of IGF1R auto-phosphorylation. Data are shown as
absorption at 450 nm (ABS 450 nm) of triplicate samples +/- Std
Error and were directly proportional to IGF1R
phosphorylation/activation.
TABLE-US-00001 Sequence Listing Heavy chain variable domain region
Light chain variable domain region GFA1
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMS QSVLTQPPSVSKGLRQTATLTCTGNSNNVG
WVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTI NQGAAWLQQHQGHPPKLLSYRNNNRPSG
SRDNAKNSLYLQMNSLRAEDTAVYYCARGHDFGG ISERFSASRSGNTASLTITGLQPEDEADYYCS
NSGYFDYWGQGTLVTVSS SEQ ID NO. 1 AWDSSLSAWVFGGXTQLTVL SEQ ID NO. 2
GFA3 QVQLVESGAEVKKPGASVKVSCKASGYTFTTYNMH
QSVLTQPASVSGSPGQSITISCTGNNRDVG WVRQAPGQGPEWMGVINPSGSSTSYAQKFQGRV
GYNYVSWFQQYPGKAPKLLIYDVSHRPSGV TMTRDTSTSTVYMQLSSLRSEDTAVYYCARWSHEA
SNRFSGSKAGNTASLTISGLQAEDEADYYCS FDIWGQGTMVTVSS SEQ ID NO. 3
SYTSSSTLVFGGGTKLTVL SEQ ID NO. 4 GFA5
EVQLVESGGGLVKPGGSLRLSCAASGFSISDYYMSW
QSVLTQPASVSGSPGQSITISCTGTSSDVGG
IRQAPGKGLEWVSYISSSSRYTNYADSVKGRFTISRD YNLVSWYQQHPGKAPKLMIFEVSQRPSGV
SAKNSLYLQMNSLRAEDTAVYYCAREGGGCNNTSC SDRFSGSKSGNTASLTVSGLQADDEANYYC
YGDGMDVWGQGTTVTVSS SEQ ID NO. 5 QSYDSSVNGWIFGGXTKLTVL SEQ ID NO. 6
GFA6 EVQLVESGGGLVQPGGSLRLSCAASGFTFSIYAMT
QAGLTQPASVSGSPGQSITISCTGTSSDVGG
WVRQAPGKGLEWVSSISGSSGYIYYADSLKGRFTISR YNYVSWYQQHPGKAPKLMIYDVSNRPSGV
DNAKNSLYLQMNSLRDEDTAVYYCARGWQGAYYG SNRFSGSKSGNTASLTISGLQAEDEADYYCS
MDVWGQGTTVTVSS SEQ ID NO. 7 SYTSSSTGVFGGGTKLTVL SEQ ID NO. 8 GFA12
QLVQSGSEVKKPGASVKVSCKASGYTFTSYYMHWV QAGLTQPASVSGSPGQSITISCTGTSSDVGG
RQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMT YNYVSWYQQHPGKAPKLMIYDVSNRPSGV
RDTSTSTVYMELSSLRSEDTAVYYCAVGGGGRYWG SNRFSGSKSGNTASLTISGLQAEDEADYYCS
QGTTVTVSS SEQ ID NO. 9 SYTSSNTLVFGGGTKLTVL SEQ ID NO. 10 GFC2
QLVQSVAEVKKPGASLTVSCTASGYTFDDYLITWVR
QSALTQPASVSGSPGQSITISCTGTSSDVGSY QAPGQGLEWLGWINTFNGKTNYAQKFQARVTMT
NLVSWYQQHPGKAPKLMIYEGSKRPSGVS RDTSTETAYLELASLTSDDTAVYYCARDYSGWYPFYL
NRFSGSKSGNTASLTISGLQAEDEADYYCSS DFWGQGTLVTVSS SEQ ID NO. 11
YTSRSTYVFGTGTKVTVL SEQ ID NO. 12 A2
QVQLVESGGGVVQPGRSLRLSCAASGFTFSRHDMY QSALTQPPSVSAAPGQKVTISCSGSSSNIGN
WVRQAPGKGLEWVAGIWYTGSKIFYADSVKGRFSI NYVSWYQQLPGTAPKLLIYDNNERPSGISN
SRDNSKNTLYLQMNSLRAEDTAVYYCAREFEAWSG
RFSGSKSGNTASLTISGLQAEDEADYYCSSYT YFGFDKWGQGTLVTVSS SEQ ID NO. 13
SSSTYVFGTGTKVTVL SEQ ID NO. 14 A11
QVQLVQSGAEVKKPGASVKVSCKASSYTFTSNGISW
SETTLTQSPAFLSATPGDKVNISCKASQDID VRQAPGQGLEWMGWINTYNGLTKYAQKLQGRLT
DDVNWYQRKPGEAAIFIIDEASNLVPGVSP MTTDTSTSTAYMELRSLRSDDTAVYYCARDRQRWL
RFSGSGYGTDFTLTINNVESEDAAYYFCLQH QGGGSGYGMDVWGQGTTVTVSS SEQ ID NO.
15 DHVPITFGQGTRLEIK SEQ ID NO. 16 B9
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYEMN QSVVTQPPSVSAAPGQKVTISCAGSTSNIG
WVRQAPGKGLEWVSYISTGDSTRSYADSVRGRFTIS NNFVSWYQQLPGTAPKLLIYDNNKRPSEIP
RDNAKNSLYLQMNSLRAEDTAVYYCARESGTWYG DRFSGSKSGTSATLGITGLQTGDEADYYCVT
GWYFDLWGRGTLVTVSS SEQ ID NO. 17 WDSSLSVVLFGGGTKVTVL SEQ ID NO. 18
B10 QVQLVQSGAEVKKPGASVKVSCKGSGYNFPTQAIH
SYELMQPSSVSVSPGQTARITCSGDLLTRRY WVRQAPGQRLEWMGWTNTANGNAKYSQKFQGR
ARWFQQKPGQAPLLIIYRDTVRPSGIPERFS
VTITRDTYASTDYMELSSLTSEDTAIYYCTRDRFTGSG
ASSSGATITLTISGAQLEDEADYYCYSATDN TYGMDVWGQGTTVTVSS SEQ ID NO. 19
NVVFGGGTKLTVL SEQ ID NO. 20 A6 QVQLVQSGAEVMKPGASVKVSC KASGYTFTSYGIS
QPVLTQPPSVSAAPGQKVTISCSGGTSNVA WVRQAPGQGLEWMGWISAYNGNTNYAQMLQG
NNYVSWYQQLPGTAPKLLIYGNSNRPSGVP RVTMTTDTSTSTAYMELRSLRSDDTAVYYCARRGLS
DRFSGSKSGTSASLAITGLQAEDEADYYCQS SYYYGMDVWGQGTTVTVSS SEQ ID NO. 21
YDTSLSGYVFGSGTKVTVL SEQ ID NO. 22 C8
QVQLVQSGAEVKKPGSSVKVSCKASGGSFNSFSISW VIWMTQSPSSLSASVGDRVTFTCQASQHIS
VRQAPGQGLEWMGGITPMFGIGDNAQKFQDRVA KYLNWYQQKPGKAPKLLIYDASNLETGVPS
ITADESMSTFYMELSNLRFEDTAMYFCAREVGGLGF
RFSGSGSATDFTLTISSLQPEDFATYYCQQSY DVWGQGTTVTVSS SEQ ID NO. 23
LTSYTFGQGTKVDIK SEQ ID NO. 24 C4
EVQLVESGGGVVQPGRSLRLSCAASRFTFSNYAMH
DIVMTQSPSSLSASVGDRVTITCRASQSISTY
WVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTI LNWFQQKPGKAPRLLIYAASNLQSGVPSRF
SRDNSKNTLYLQMNSLRAEDTAVYYCAREVLEYYYD
SGGGSGTDFTLTINSLQPEDFATYYCQQSYS SSGAFDIWGQGTMVTVSS SEQ ID NO. 25
TPFTFGPGTKVDIK SEQ ID NO. 26 E2 QVQLVQSGAEVKKPGTSVKVSCKASGGAFNRFPIS
LPVLTQPASVSGSPGQSITISCTGTSIDVASY WVRQAPGQGLEWMGWISPNGGNTNYAQKFQGR
NLVSWYQQHPGKAPKLMIYDVSNRPSGVS VTMTRDTSINTAYMEVSSLTSDDTAVYYCTQGRVAF
TRFSGSKSGNTASLTISGLQAEDEADYYCISR VWGQGTLVTVSS SEQ ID NO. 27
ANSNTLYVFGTGTKVTVL SEQ ID NO. 28 B3
QVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYAMH VIWMTQSPSSLSASVGDRVTITCRATQSIST
WVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTI YLNWYQQKPGKAPNLLIYAASSLQSGVPSR
SRDNSKNTLYLQMNSLRAEDTAVYYCARELWYGEG
FSGSGSGTDFTLTISSLQPEDFATYYCQQSYR FDPWGQGTLVTVSS SEQ ID NO. 29
TPGTFGQGTKVDIK SEQ ID NO. 30 D12
QVQLVESGAEVKKPGASVKVSCKASGYTFTTYNMH QPVLTQPPSVSAAPGQKVTISCSGSSSNIGN
WVRQAPGQGPEWMGVINPSGSSTSYAQKFQGRV VLTQPASVSGSPGQSITISCTGNNRDVGGY
TMTRDTSTSTVYMQLSSLRSEDTAVYYCARWSHEA NYVSWFQQYPGKAPKLLIYDVSHRPSGVSN
FDIWGQGTMVTVSS SEQ ID NO. 31 RFSGSKAGNTASLTISGLQAEDEADYYCSSY
TSSSTLVFGGGTKLTVL SEQ ID NO. 32
Sequence CWU 1
1
321122PRTHomo sapians 1Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asn Ile Lys Gln
Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly His Asp Phe Gly Gly Asn Ser Gly Tyr Phe Asp Tyr Trp
100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
2110PRTHomo sapiansmisc_feature(104)..(104)Xaa can be any naturally
occurring amino acid 2Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser
Lys Gly Leu Arg Gln 1 5 10 15 Thr Ala Thr Leu Thr Cys Thr Gly Asn
Ser Asn Asn Val Gly Asn Gln 20 25 30 Gly Ala Ala Trp Leu Gln Gln
His Gln Gly His Pro Pro Lys Leu Leu 35 40 45 Ser Tyr Arg Asn Asn
Asn Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser 50 55 60 Ala Ser Arg
Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Leu Gln 65 70 75 80 Pro
Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ala Trp Asp Ser Ser Leu 85 90
95 Ser Ala Trp Val Phe Gly Gly Xaa Thr Gln Leu Thr Val Leu 100 105
110 3117PRTHomo sapians 3Gln Val Gln Leu Val Glu Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Thr Tyr 20 25 30 Asn Met His Trp Val Arg
Gln Ala Pro Gly Gln Gly Pro Glu Trp Met 35 40 45 Gly Val Ile Asn
Pro Ser Gly Ser Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly
Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80
Met Gln Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Trp Ser His Glu Ala Phe Asp Ile Trp Gly Gln Gly Thr
Met 100 105 110 Val Thr Val Ser Ser 115 4110PRTHomo sapians 4Gln
Ser Val Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10
15 Ser Ile Thr Ile Ser Cys Thr Gly Asn Asn Arg Asp Val Gly Gly Tyr
20 25 30 Asn Tyr Val Ser Trp Phe Gln Gln Tyr Pro Gly Lys Ala Pro
Lys Leu 35 40 45 Leu Ile Tyr Asp Val Ser His Arg Pro Ser Gly Val
Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ala Gly Asn Thr Ala Ser
Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr
Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90 95 Ser Thr Leu Val Phe Gly
Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 5126PRTHomo sapians
5Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Ile Ser Asp
Tyr 20 25 30 Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Ser Arg Tyr Thr Asn
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Ser Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Gly
Gly Cys Asn Asn Thr Ser Cys Tyr Gly Asp Gly 100 105 110 Met Asp Val
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 6111PRTHomo
sapiansmisc_feature(105)..(105)Xaa can be any naturally occurring
amino acid 6Gln Ser Val Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro
Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp
Val Gly Gly Tyr 20 25 30 Asn Leu Val Ser Trp Tyr Gln Gln His Pro
Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Phe Glu Val Ser Gln Arg
Pro Ser Gly Val Ser Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly
Asn Thr Ala Ser Leu Thr Val Ser Gly Leu 65 70 75 80 Gln Ala Asp Asp
Glu Ala Asn Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser 85 90 95 Val Asn
Gly Trp Ile Phe Gly Gly Xaa Thr Lys Leu Thr Val Leu 100 105 110
7120PRTHomo sapians 7Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Ala Met Thr Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly
Ser Ser Gly Tyr Ile Tyr Tyr Ala Asp Ser Leu 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Trp Gln Gly Ala Tyr Tyr Gly Met Asp Val Trp Gly Gln
100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 8110PRTHomo
sapians 8Gln Ala Gly Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro
Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp
Val Gly Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro
Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Asp Val Ser Asn Arg
Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly
Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp
Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90 95 Ser Thr
Gly Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110
9113PRTHomo sapians 9Gln Leu Val Gln Ser Gly Ser Glu Val Lys Lys
Pro Gly Ala Ser Val 1 5 10 15 Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Ser Tyr Tyr Met 20 25 30 His Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met Gly Ile 35 40 45 Ile Asn Pro Ser Gly
Gly Ser Thr Ser Tyr Ala Gln Lys Phe Gln Gly 50 55 60 Arg Val Thr
Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu 65 70 75 80 Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Val 85 90
95 Gly Gly Gly Gly Arg Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser
100 105 110 Ser 10110PRTHomo sapians 10Gln Ala Gly Leu Thr Gln Pro
Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser
Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Tyr Val
Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met
Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55
60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr
Ser Ser 85 90 95 Asn Thr Leu Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu 100 105 110 11119PRTHomo sapians 11Gln Leu Val Gln Ser Val
Ala Glu Val Lys Lys Pro Gly Ala Ser Leu 1 5 10 15 Thr Val Ser Cys
Thr Ala Ser Gly Tyr Thr Phe Asp Asp Tyr Leu Ile 20 25 30 Thr Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Leu Gly Trp 35 40 45
Ile Asn Thr Phe Asn Gly Lys Thr Asn Tyr Ala Gln Lys Phe Gln Ala 50
55 60 Arg Val Thr Met Thr Arg Asp Thr Ser Thr Glu Thr Ala Tyr Leu
Glu 65 70 75 80 Leu Ala Ser Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr
Cys Ala Arg 85 90 95 Asp Tyr Ser Gly Trp Tyr Pro Phe Tyr Leu Asp
Phe Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
12110PRTHomo sapians 12Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser
Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr
Ser Ser Asp Val Gly Ser Tyr 20 25 30 Asn Leu Val Ser Trp Tyr Gln
Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Glu Gly
Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser
Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln
Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Arg 85 90
95 Ser Thr Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu 100 105
110 13122PRTHomo sapians 13Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Arg His 20 25 30 Asp Met Tyr Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly Ile Trp
Tyr Thr Gly Ser Lys Ile Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Ser Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Glu Phe Glu Ala Trp Ser Gly Tyr Phe Gly Phe Asp Lys
Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
14109PRTHomo sapians 14Gln Ser Ala Leu Thr Gln Pro Pro Ser Val Ser
Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Ser Gly Ser
Ser Ser Asn Ile Gly Asn Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln
Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Asn
Glu Arg Pro Ser Gly Ile Ser Asn Arg Phe Ser 50 55 60 Gly Ser Lys
Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln 65 70 75 80 Ala
Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser Ser 85 90
95 Thr Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu 100 105
15126PRTHomo sapians 15Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Ser Tyr Thr Phe Thr Ser Asn 20 25 30 Gly Ile Ser Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr
Tyr Asn Gly Leu Thr Lys Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg
Leu Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Asp Arg Gln Arg Trp Leu Gln Gly Gly Gly Ser Gly Tyr Gly
100 105 110 Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 125 16108PRTHomo sapians 16Ser Glu Thr Thr Leu Thr Gln Ser
Pro Ala Phe Leu Ser Ala Thr Pro 1 5 10 15 Gly Asp Lys Val Asn Ile
Ser Cys Lys Ala Ser Gln Asp Ile Asp Asp 20 25 30 Asp Val Asn Trp
Tyr Gln Arg Lys Pro Gly Glu Ala Ala Ile Phe Ile 35 40 45 Ile Asp
Glu Ala Ser Asn Leu Val Pro Gly Val Ser Pro Arg Phe Ser 50 55 60
Gly Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Asn Asn Val Glu 65
70 75 80 Ser Glu Asp Ala Ala Tyr Tyr Phe Cys Leu Gln His Asp His
Val Pro 85 90 95 Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
100 105 17122PRTHomo sapians 17Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Glu Met Asn Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile
Ser Thr Gly Asp Ser Thr Arg Ser Tyr Ala Asp Ser Val 50 55 60 Arg
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Glu Ser Gly Thr Trp Tyr Gly Gly Trp Tyr Phe
Asp Leu Trp 100 105 110 Gly Arg Gly Thr Leu Val Thr Val Ser Ser 115
120 18110PRTHomo sapians 18Gln Ser Val Val Thr Gln Pro Pro Ser Val
Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Ala Gly
Ser Thr Ser Asn Ile Gly Asn Asn 20 25 30 Phe Val Ser Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn
Asn Lys Arg Pro Ser Glu Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser
Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln 65 70 75 80
Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Val Thr Trp Asp Ser Ser Leu 85
90 95 Ser Val Val Leu Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100
105 110 19122PRTHomo sapians 19Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys
Gly Ser Gly Tyr Asn Phe Pro Thr Gln 20 25 30 Ala Ile His Trp Val
Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Thr
Asn Thr Ala Asn Gly Asn Ala Lys Tyr Ser Gln Lys Phe 50 55 60 Gln
Gly Arg Val Thr Ile Thr Arg Asp Thr Tyr Ala Ser Thr Asp Tyr 65 70
75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Ile Tyr Tyr
Cys 85 90 95 Thr Arg Asp Arg Phe Thr Gly Ser Gly Thr Tyr Gly Met
Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115
120 20106PRTHomo sapians 20Ser Tyr Glu Leu Met Gln Pro Ser Ser Val
Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Ser Gly
Asp Leu Leu Thr Arg Arg Tyr Ala
20 25 30 Arg Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Leu Leu Ile
Ile Tyr 35 40 45 Arg Asp Thr Val Arg Pro Ser Gly Ile Pro Glu Arg
Phe Ser Ala Ser 50 55 60 Ser Ser Gly Ala Thr Ile Thr Leu Thr Ile
Ser Gly Ala Gln Leu Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Tyr
Ser Ala Thr Asp Asn Asn Val Val 85 90 95 Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu 100 105 21121PRTHomo sapians 21Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Met Lys Pro Gly Ala 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly
Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Met Leu
50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr
Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Gly Leu Ser Ser Tyr Tyr Tyr
Gly Met Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser
Ser 115 120 22110PRTHomo sapians 22Gln Pro Val Leu Thr Gln Pro Pro
Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys
Ser Gly Gly Thr Ser Asn Val Ala Asn Asn 20 25 30 Tyr Val Ser Trp
Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr
Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60
Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln 65
70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Thr
Ser Leu 85 90 95 Ser Gly Tyr Val Phe Gly Ser Gly Thr Lys Val Thr
Val Leu 100 105 110 23118PRTHomo sapians 23Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Gly Ser Phe Asn Ser Phe 20 25 30 Ser Ile
Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Gly Ile Thr Pro Met Phe Gly Ile Gly Asp Asn Ala Gln Lys Phe 50
55 60 Gln Asp Arg Val Ala Ile Thr Ala Asp Glu Ser Met Ser Thr Phe
Tyr 65 70 75 80 Met Glu Leu Ser Asn Leu Arg Phe Glu Asp Thr Ala Met
Tyr Phe Cys 85 90 95 Ala Arg Glu Val Gly Gly Leu Gly Phe Asp Val
Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115
24107PRTHomo sapians 24Val Ile Trp Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Phe Thr Cys Gln Ala
Ser Gln His Ile Ser Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn
Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Leu Thr Ser Tyr 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys 100 105 25124PRTHomo
sapians 25Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro
Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Phe Thr
Phe Ser Asn Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser
Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Glu Val Leu Glu Tyr Tyr Tyr Asp Ser Ser Gly Ala Phe Asp 100 105 110
Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120
26107PRTHomo sapians 26Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Ser Thr Tyr 20 25 30 Leu Asn Trp Phe Gln Gln Lys
Pro Gly Lys Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Asn
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Gly Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Phe 85 90
95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100 105 27115PRTHomo
sapians 27Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Thr 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Ala
Phe Asn Arg Phe 20 25 30 Pro Ile Ser Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Pro Asn Gly Gly
Asn Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Ile Asn Thr Ala Tyr 65 70 75 80 Met Glu Val Ser
Ser Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Gln
Gly Arg Val Ala Phe Val Trp Gly Gln Gly Thr Leu Val Thr 100 105 110
Val Ser Ser 115 28111PRTHomo sapians 28Leu Pro Val Leu Thr Gln Pro
Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser
Cys Thr Gly Thr Ser Ile Asp Val Ala Ser Tyr 20 25 30 Asn Leu Val
Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met
Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Thr Arg Phe 50 55
60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ile Ser Arg Ala
Asn Ser 85 90 95 Asn Thr Leu Tyr Val Phe Gly Thr Gly Thr Lys Val
Thr Val Leu 100 105 110 29119PRTHomo sapians 29Gln Val Gln Leu Val
Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Leu Trp Tyr Gly Glu Gly Phe
Asp Pro Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
30107PRTHomo sapians 30Val Ile Trp Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Thr Gln Ser Ile Ser Thr Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Asn Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Arg Thr Pro Gly 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys 100 105 31117PRTHomo
sapians 31Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Thr Tyr 20 25 30 Asn Met His Trp Val Arg Gln Ala Pro Gly
Gln Gly Pro Glu Trp Met 35 40 45 Gly Val Ile Asn Pro Ser Gly Ser
Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met
Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Gln Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Trp Ser His Glu Ala Phe Asp Ile Trp Gly Gln Gly Thr Met 100 105 110
Val Thr Val Ser Ser 115 32139PRTHomo sapians 32Gln Pro Val Leu Thr
Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr
Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Val 20 25 30 Leu
Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln Ser Ile Thr 35 40
45 Ile Ser Cys Thr Gly Asn Asn Arg Asp Val Gly Gly Tyr Asn Tyr Val
50 55 60 Ser Trp Phe Gln Gln Tyr Pro Gly Lys Ala Pro Lys Leu Leu
Ile Tyr 65 70 75 80 Asp Val Ser His Arg Pro Ser Gly Val Ser Asn Arg
Phe Ser Gly Ser 85 90 95 Lys Ala Gly Asn Thr Ala Ser Leu Thr Ile
Ser Gly Leu Gln Ala Glu 100 105 110 Asp Glu Ala Asp Tyr Tyr Cys Ser
Ser Tyr Thr Ser Ser Ser Thr Leu 115 120 125 Val Phe Gly Gly Gly Thr
Lys Leu Thr Val Leu 130 135
* * * * *
References